Dystrophin在不同类型肌营养不良症中的变化及诊断价值

目的研究dystrophin在不同类型肌营养不良症中的变化及分型诊断价值.方法用抗dystrophin抗体对107例肌营养不良症患者肌组织标本行免疫组织化学分析.结果Duchenne型肌营养不良(DMD)患者肌细胞膜上无显色,Becker型肌营养不良(BMD)患者肌细胞膜上显色浅淡、不连续或呈斑片状.肢带型肌营养不良(LGMD)患者肌细胞膜上染色正常.结论dystrophin免疫组化染色对于年龄较小临床不易区分的DMD/BMD患者,可区分开来,以早期预测功能影响程度.该方法也有助于区分临床表现相似的成年散发BMD和LGMD患者,对于正确地进行遗传咨询具有重要意义.     

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     

Dystrophinopathies in females

Various laboratory tests were performed to establish carriership in 24 familial and sporadic carriers of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). The activity of creatine kinase was in all females but one, very high and significantly higher in isolated carriers; quantitative EMG indicated myopathic changes, muscle biopsies revealed different degrees of changes--from a variability of muscle fibers size and central nuclei to severe dystrophic features. Immunohistochemical evaluation of dystrophin revealed, in all females but one, mosaic pattern of staining--a mixture of dystrophin-positive and dystrophin-negative fibers, the latter consist 15-30% of all fibers. Quantitative evaluation of dystrophin showed a reduced abundance with normal or abnormal molecular weight. The abnormalities were more expressed in sporadic cases. The detection of sporadic carriers, particularly the non-manifesting clinical, is a very important progress--it permits the correct diagnosis (before, these females were diagnosed as limb girdle muscle dystrophy (LGMD) and supply them with the benefit of genetic counselling, which also requires some modification.     

Limb girdle muscular dystrophy: A pathological and immunohistochemical reevaluation

Ninety-seven muscle biopsies from 81 limb girdle muscular dystrophy (LGMD) patients [32 autosomal recessive (AR), 15 autosomal dominant (AD), 34 sporadic] were morphologically reevaluated. Sarcoglycan analysis was done in 37 available muscle biopsies of AR and sporadic patients. Chi-square tests were used to analyze the relation between abnormalities in AR/sporadic versus AD cases. Eighty percent of the muscle biopsies showed a predominantly dystrophic pattern, 20% showed myopathic changes, and 17% of these also had neurogenic changes. Muscle histology was not significantly different between AR/sporadic and AD LGMD; however, the observed abnormalities were more pronounced in the AR/sporadic group. Collections of inflammatory cells were observed in 25% and 10% of the AR/sporadic and AD group, respectively. Sarcoglycanopathy was diagnosed in 25% of the AR and sporadic patients of the 37 families tested. We conclude that the histological picture of AR/sporadic and AD LGMD is essentially the same, and sarcoglycanopathy constitutes an important part of the AR/sporadic patients. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:584–590, 1998.     

Episodes of exercise-induced dark urine and myalgia in LGMD 2I

Lindberg C, Sixt C, Oldfors A. Episodes of exercise‐induced dark urine and myalgia in LGMD 2I.
Acta Neurol Scand: 2012: 125: 285–287.
© 2011 John Wiley & Sons A/S. Background – Mutations in the fukutin‐related protein gene FKRP (MIM *606596) cause a form of congenital muscular dystrophy (MDC1C) and also limb girdle muscular dystrophy type 2I (LGMD2I). Exercise‐induced myoglobinuria, frequently occurring in metabolic myopathies, has been described in Becker muscular dystrophy and in a few cases of LGMD. Objectives – To describe that episodes with myoglobinuria, often associated with exercise‐induced myalgia, may be common and a presenting symptom in patients with LGMD2I. Methods – Data on episodes of suspected myoglobinuria and myalgia were collected from the patient records on 14 patients with a diagnosis of LGMDI. Results – Five LGMD2I patients reported recurrent episodes of dark urine and myalgia after exercise, and in three of them, this was the only symptom for several years. Conclusions – We conclude that episodes compatible with exercise‐induced myoglobinuria may be frequent in LGMD2I.     

Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy

Within a group of 76 sporadic/autosomal recessive limb girdle muscular dystrophy (LGMD) patients we tried to identify those with LGMD type 2C-E. Muscle biopsy specimens of 40 index patients, who had 22 affected sibs, were analyzed immuno-histochemically for the presence of three subunits: α-, β-, and γ,-sarcoglycans. Abnormal sarcoglycan expression was established in eight patients, with six affected sibs. In one patient γ-sarcoglycan was absent, and both α- and β-sarcoglycans were reduced. In the remaining seven patients γ-sarcoglycan was (slightly) reduced, and α- and β-sarcoglycans were absent or reduced. By DNA sequencing mutations were detected in one of the three sarcoglycan genes in all eight cases. Three patients had mutations in the α-, three in the β-, and two in the γ-sarcoglycan gene. The patients with sarcoglycanopathy comprised the more severely affected cases (P=0.04). In conclusion, sarcoglycanopathy was identified in 23% (14/62) of the autosomal recessive LGMD patients. Received: 11 November 1999, Received in revised form: 27 January 2000, Accepted: 29 February 2000     

Pathological changes of the myonuclear fibrous lamina and internal nuclear membrane in two cases of autosomal dominant limb-girdle muscular dystrophy with atrioventricular conduction disturbance (LGMD1B)

Mutations in the lamin A/C gene have been reported in a variety of disorders including autosomal dominant Emery-Dreifuss muscular dystrophy and autosomal dominant limb girdle muscular dystrophy with cardiac conduction block or limb girdle muscular dystrophy type 1B (LGMD1B). However, how these mutations are involved in developing these diseases is not known. We examined morphological changes of the skeletal muscle in two cases of LGMD1B in a family, directing our attention to the nuclear envelope and its underlying structures where lamin A/C is located. Although conventional fluorescence microscope revealed no discernible abnormality in the distribution of emerin and lamin A/C, a serial multi-layer scanning with confocal laser scanning microscope showed an attenuated and uneven distribution of lamin A/C. Furthermore, under an electron microscope, the nuclear fibrous lamina and inner nuclear membrane were relatively indistinct compared to controls. These changes in the myonuclei may be related to pathomechanisms of the present cases.     

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.     

A new mutation of the fukutin gene causing late-onset limb girdle muscular dystrophy

Defects in glycosylations of α-dystroglycan are associated with mutations in several genes, including the fukutin gene (FKTN). Hypoglycosylation of α-dystroglycan results in several forms of muscular dystrophy with variable phenotype. Outside Japan, the prevalence of muscular dystrophies related to aberrations of FKTN is rare, with only eight reported cases of limb girdle phenotype (LGMD2M). We describe the mildest affected patient outside Japan with genetically confirmed LGMD2M and onset of symptoms at age 14. She was brought to medical attention at age 12, not because of muscle weakness, but due to episodes of tachycardia caused by Wolff–Parkinson–White syndrome. On examination, she had rigid spine syndrome, a typical limb girdle dystrophy pattern of muscle weakness, cardiomyopathy, and serum CK levels >2000 IU/L (normal <150 IU/L). A homozygous, novel c.917A>G; p.Y306C mutation in the FKTN gene was found. The case confirms FKTN mutations as a cause of LGMD2M without mental retardation and expands the phenotypic spectrum for LGMD2M to include cardiomyopathy and rigid spine syndrome in the mildest affected non-Japanese patient reported so far.     

LAMA2‐related myopathy: Frequency among congenital and limb‐girdle muscular dystrophies

Introduction: Muscular dystrophy caused by LAMA2‐gene mutations is an autosomal recessive disease typically presenting as a severe, early‐onset congenital muscular dystrophy (CMD). However, milder cases with a limb‐girdle type muscular dystrophy (LGMD) have been described. Methods: In this study, we assessed the frequency and phenotypic spectrum of LAMA2‐related muscular dystrophy in CMD (n = 18) and LGMD2 (n = 128) cohorts identified in the last 15 years in eastern Denmark. The medical history, brain‐MRI, muscle pathology, muscle laminin‐α2 expression, and genetic analyses were assessed. Results: Molecular genetics revealed 2 pathogenic LAMA2 mutations in 5 of 18 CMD and 3 of 128 LGMD patients, corresponding to a LAMA2‐mutation frequency of 28% in the CMD and 2.3% in the LGMD cohorts, respectively. Conclusions: This study demonstrates a wide clinical spectrum of LAMA2‐related muscular dystrophy and its prevalence in an LGMD2 cohort, which indicates that LAMA2 muscular dystrophy should be included in the LGMD2 nomenclature. Muscle Nerve 52: 547–553, 2015     

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.     

Limb girdle muscular dystrophy: Description of a phenotype

The phenotype is reported of 20 patients with autosomal recessive or sporadic, pelvifemoral limb girdle muscular dystrophy (LGMD). Selective wasting of muscles was observed at the moderately advanced stage of illness. The pattern of weakness was uniform. Attention to clinical detail allowed the identification of a phenotype different from a hypothetical scheme of LGMD based on previous literature, and other causes of limb girdle weakness. These patients may represent yet another nosologic entity within the autosomal recessive dystrophies; molecular genetic studies are awaited. A limited magnetic resonance imaging (MRI) study of muscle was of little consequence. Although additional detail was obtained, no pathognomonic distribution of the dystrophic process was observed; interindividual variation existed even among closely matched siblings. The severity of MRI signal change did not consistently correlate with the degree of weakness in an individual. When a diagnosis is uncertain, however, the added detail may be useful. © 1994 John Wiley & Sons, Inc.     

Diagnostic value of muscle MRI in differentiating LGMD2I from other LGMDs

Mutations in the fukutin–related protein (FKRP) have recently been demonstrated to cause limb girdle muscular dystrophy type 2I (LGMD2I), one of the most common forms of the autosomal recessive LGMDs in Europe. We performed a systematic clinical and muscle MRI assessment in 6 LGMD2I patients and compared these findings with those of 14 patients with genetically confirmed diagnosis of other forms of autosomal recessive LGMDs or dystrophinopathies. All LGMD2I patients had a characteristic clinical phenotype with predominant weakness of hip flexion and adduction, knee flexion and ankle dorsiflexion. These findings were also mirrored on MRI of the lower extremities which demonstrated marked signal changes in the adductor muscles, the posterior thigh and posterior calf muscles. This characteristic clinical and MRI phenotype was also seen in LGMD2A. However, in LGMD2A there was a selective involvement of the medial gastrocnemius and soleus muscle in the lower legs which was not seen in LGMD2I. The pattern in LGMD2A and LGMD2I were clearly different from the one seen in alpha–sarcoglycanopathy and dystrophinopathy type Becker which showed marked signal abnormalities in the anterior thigh muscles. Our results indicate that muscular MRI is a powerful tool for differentiating LGMD2I from other forms of autosomal recessive LGMDs and dystrophinopathies.     

Dystrophin immunostaining in muscles from patients with different types of muscular dystrophy: a Brazilian study.

The localization of the protein dystrophin was studied using the immunofluorescence method, in muscle biopsies from 74 patients affected by different types of muscular dystrophy and 4 normal controls. In 15 patients with limb-girdle muscular dystrophy (LGMD) the pattern was indistinguishable from normal. Among 42 Duchenne patients (DMD), 3 were totally negative and 39 showed a variable proportion (4-30%) of partially labelled fibers. With one exception 17 Becker dystrophy patients (BMD), showed a positive sarcolemmal reaction. A diffuse reaction inside the fibers, which was not observed in normal controls, was seen in the majority of DMD and also in some of the BMD patients. Based on these observations it is suggested that in DMD, a small quantity of protein is still present or there is a cross-reaction with other proteins which share some homology with dystrophin. The present results suggest that it is possible to make a differential diagnosis between DMD and BMD through dystrophin immunohistochemistry. However, to distinguish between patients with BMD and LGMD phenotypes, or DMD and outliers, complementary immunoblot studies and quantitative determination of dystrophin are necessary.     

Dystrophin-associated protein abnormalities in dystrophin-deficient muscle fibers from symptomatic and asymptomatic Duchenne/Becker muscular dystrophy carriers

The absence of dystrophin in muscle fibers is associated with a major reduction in dystrophin-associated proteins (DAPs) and disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. We investigated the expression of the DAPs β-dystroglycan, α-sarcoglycan, γ-sarcoglycan and syntrophin as well as utrophin in the muscles of 13 Duchenne muscular dystrophy (DMD) carriers (with variable percentages of dystrophin-deficient fibers and with a range of clinical symptoms), 2 Becker muscular dystrophy (BMD) carriers (expressing a highly truncated protein in some fibers), 2 girls with a DMD-like phenotype, and 11 BMD carriers with almost normal dystrophin expression (reduced or patchy distribution in a few fibers only and rare dystrophin-deficient fibers). DAPs were highly reduced in all fibers lacking dystrophin in the DMD carriers, but were almost normal in the dystrophin-deficient fibers of the 2 BMD carriers with highly truncated dystrophin. In the 11 BMD carriers with nearly normal dystrophin, the few fibers with reduced or patchy dystrophin immunostaining also showed reduced DAP expression in correlation with dystrophin expression. Immunoblot for β-dystroglycan and α-sarcoglycan confirmed the immunohistochemical findings. Utrophin expression was slightly increased in a proportion of fibers in the DMD and BMD carriers with dystrophin mosaicism. We found no correlation between utrophin expression and DAP expression. We conclude that absence or reduction of dystrophin in muscle fibers of DMD and BMD carriers causes a reduction of DAPs in the same fibers, as observed in DMD and BMD patients, while utrophin does not seem to play a role in DAP expression in adult muscle. Received: 11 January 1996 / Revised, accepted: 16 April 1996     

Delayed expression of dystrophin on regenerating muscle from two siblings with Becker muscular dystrophy

We present here a unique expression of dystrophin on biopsied muscle from 2 siblings with Becker muscular dystrophy (BMD). They had neither muscle weakness nor atrophy. Clustered dystrophin-deficient fibers were constituted to regenerating basophilic fibers (mainly type 2C fiber) based on histochemical stainings. We speculate that the developmental delay in the expression of dystrophin is a characteristics finding in regenerating fibers from asymptomatic and young BMD patients, such as the siblings in this report.     

ANO5 mutations in the Dutch limb girdle muscular dystrophy population

A Dutch cohort of 105 limb girdle muscular dystrophy (LGMD) patients were subject to subsequent genetic investigations. In half the families a causative mutation was found. Recently mutations were identified in ANO5 causing LGMD2L and Miyoshi-like myopathy (MMD3), but could also be found in patients with hyperCKemia only. Therefore, we analysed the index cases of the remaining 31 as yet undiagnosed families from our previously described cohort of LGMD patients for the presence of ANO5 mutations. Detailed history and neurological examination were available for all patients. Serum creatine kinase (CK) activity, skeletal muscle computed tomography (CT) and cardiological investigations were performed. Mutations in ANO5 were found in 16% of the families: 11 index patients and two sibs, eight males and five females. The founder mutation c.191dupA was present in 8 out of 13 patients. Ten different pathogenic mutations were identified of which seven were novel: five missense and two splice site mutations. The age of these patients ranged from 26 to 69 years and the age of onset varied from 21 to 57 years. Symptoms at onset were related to proximal leg weakness. The weakness was slowly progressive. Calf hypertrophy was present in three patients. Males were more severely affected than females. Serum CK activity was highly elevated in the early stage of disease and moderately increased in later stages. Muscle biopsy showed predominantly dystrophic changes. One patient had hypertrophic cardiomyopathy, two others had intraventricular septum thickening.     

The abnormal expression of utrophin in Duchenne and Becker muscular dystrophy is age related

J. Taylor, F. Muntoni, V. Dubowitz and C. A. Sewry (1997) Neuropathology and Applied Neurobiology 23, 399–405 The abnormal expression of utrophin in Duchenne and Becker muscular dystrophy is age related Utrophin is a 395 kDa protein with considerable homology to dystrophin. It is highly expressed in the sarcolemma of normal fetal muscle fibres but is confined to neuromuscular and myotendinous junctions, and blood vessels in adult muscle. Sarcolemmal expression occurs on regenerating fibres, irrespective of the disease, and is also seen on mature fibres in Duchenne and Becker muscular dystrophies (DMD, BMD), and inflammatory myopathies. The reasons for the abnormal expression in DMD and BMD are unclear. We have studied this expression of utrophin immunocytochemically on mature fibres in 42 cases of DMD and BMD, aged 3 months–24 years of age. All cases had some mature fibres, with no detectable fetal myosin, that showed sarcolemmal expression of utrophin. The number of these fibres and the intensity of fluorescence was low in young cases before the age of 2 years and increased with age. The fluorescence was graded on a scale of 0 to ++++ and there were significantly more cases under 2 years of age (10/12) with a grading of utrophin of only +, compared with those over 2 years (4/30, P < 0.001). Some revertant fibres, but not all, expressed utrophin and dystrophin. Our data show that the abnormal expression of utrophin on mature muscle fibres in DMD and BMD is not a continuation of the expression that occurs in fetal or regenerating muscle, but is a secondary event caused by unknown factors. The immunocytochemical intensity of utrophin is variable between cases and there is no correlation with clinical severity. As all cases studied had some expression of utrophin on mature fibres, this may be a useful additional tool for distinguishing BMD from other dystrophies, especially in cases with minimal abnormalities in dystrophin expression and/or no detectable mutation in the gene.     

Asian patients with limb girdle muscular dystrophy 2I (LGMD2I)

Limb girdle muscular dystrophy type 2I (LGMD2I) is caused by defects in the fukutin-related protein (FKRP) gene. In most Caucasian patients with LGMD2I, the condition is associated with a missense mutation - c.826C>A (p.Leu276Ile). We describe two Chinese brothers with progressive shoulder and pelvic muscle weakness. They had muscle stiffness and myalgia after exercise, but lacked obvious hypertrophy of the calves. Muscle biopsy showed dystrophic features with many rimmed vacuoles in the fibers. Immunohistochemistry and immunoblot analyses revealed reductions of alpha-(??)-dystroglycan (VIA4-1) and laminin-??2 (80-kDa C-terminal and 300-kDa N-terminal). Two novel heterozygous mutations (c.208T>A and c.1030G>T) in the FKRP gene were identified in these patients. In addition, we summarise the clinical features of patients with LGMD2I in the Asian region. Our findings might indicate that the pathogenic FKRP mutations in Asian patients with LGMD2I are sporadic compound heterozygous mutations rather than the hot-spot c.826C>A mutation seen in Caucasian populations.     

Becker muscular dystrophy: detection of unusual disease courses by combined approach to dystrophin analysis.

The rapid progress of research on the structure of the dystrophin gene has enormously increased our understanding of the molecular basis of Duchenne (DMD) and Becker (BMD) muscular dystrophy. Apart from "classical" clinical presentations, asymptomatic or only mildly affected individuals with deletions in the dystrophin gene have now been reported. We describe two families which were initially classified as metabolic myopathies, until the diagnosis of atypical BMD was established after dystrophin analysis at the protein and DNA level. A modern diagnostic approach to myopathies should, therefore, not only include morphological and biochemical investigations, but also be extended to the analysis of the dystrophin gene.