Two siblings with limb-girdle muscular dystrophy type 2E responsive to deflazacort

Two siblings were evaluated for progressive proximal weakness and elevated creatine kinase. Immunohistochemical staining in the brother’s muscle biopsy showed near absence of all four sarcoglycan subunits. Clinical progression prompted a trial of deflazacort in both siblings. At 22 months of drug therapy, both patients have stable or improved strength testing. Further analysis on the muscle biopsy revealed homozygous β-sarcoglycan gene mutation (S114F), consistent with the limb-girdle muscular dystrophy type 2E (LGME 2E). Despite the severe phenotype, deflazacort has a beneficial effect on slowing disease progression in LGME 2E similar to that seen in Duchenne muscular dystrophy.     

A new locus for autosomal recessive limb-girdle muscular dystrophy in a large consanguineous Tunisian family maps to chromosome 19q13.3

Autosomal recessive limb-girdle muscular dystrophies represent a genetically heterogeneous group of diseases characterized by a progressive involvement of skeletal muscles. They show a wide spectrum of clinical courses, varying from very mild to severe. Eight loci responsible for autosomal recessive limb-girdle muscular dystrophies have been mapped and six defective genes identified. In this study, we report the clinical data, muscle biopsy findings and results of genetic linkage analysis in a large consanguineous Tunisian family with 13 individuals suffering from autosomal recessive limb-girdle muscular dystrophy. Clinical features include variable age of onset, proximal limb muscle weakness and wasting predominantly affecting the pelvic girdle, and variable course between siblings. CK rate was usually high in younger patients. Muscle biopsy showed dystrophic changes with normal expression of dystrophin and various proteins of the dystrophin-associated protein complex (sarcoglycan sub-units, dystroglycan, and sarcospan). Genetic linkage analysis excluded the known limb-girdle muscular dystrophies loci as well as ten additional candidate genes. A maximum LOD score of 4.36 at θ=0.00 was obtained with marker D19S606, mapping this new form of autosomal recessive limb-girdle muscular dystrophy to chromosome 19q13.3.     

Mutations in the δ-sarcoglycan gene are a rare cause of autosomal recessive limb-girdle muscular dystrophy (LGMD2)

The dystrophin-based membrane cytoskeleton of muscle fibers has emerged as a critical multiprotein complex which seems to impart structural integrity on the muscle fiber plasma membrane. Deficiency of dystrophin causes the most common types of muscular dystrophy, Duchenne and Becker muscular dystrophies. Muscular dystrophy patients showing normal dystrophin protein and gene analysis are generally isolated cases with a presumed autosomal recessive inheritance pattern (limb-girdle muscular dystrophy). Recently, linkage and candidate gene analyses have shown that some cases of limb-girdle muscular dystrophy can be caused by deficiency of other components of the dystrophin membrane cytoskeleton. The most recently identified component, δ-sarcoglycan deficiency occurred in other world populations, to identify the range of mutations and clinical phenotypes, and to test for the biochemical consequences of δ-sarcoglycan gene mutations, we studied Duchenne-like and limb-girdle muscular dystrophy patients who we had previously shown not to exhibit gene mutations of dystrophin, α-, β-, or γ-sarcoglycan for δ-sarcoglycan mutations (n = 54). We identified two American patients with novel nonsense mutations of δ-sarcoglycan (W30X, R165X). One was apparently homozygous, and we show likely consanguinity through homozygosity for 13 microsatellite loci covering a 38 cM region of chromosome 5. The second was heterozygous. Both were girls who showed clinical symptoms consistent with Duchenne muscular dystrophy in males. Our data shows that δ-sarcoglycan deficiency occurs in other world populations, and that most or all patients show a deficiency of the entire sarcoglycan complex, adding support to the hypothesis that these proteins function as a tetrameric unit. Received January 1, 1997; Revised and Accepted January 15, 1997     

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.     

The beta-delta-core of sarcoglycan is essential for deposition at the plasma membrane

Mutations of any of the sarcoglycan complex subunits (alpha, beta, delta, and gamma) cause limb-girdle muscular dystrophy. Furthermore, individual mutations lead to a reduction or loss of all other members of the complex. In some cases of limb-girdle muscular dystrophies, however, residual sarcoglycan expression has been documented. Therefore, in this study we tested the hypothesis that formation of specific sarcoglycan subcomplexes is crucial for plasma membrane deposition. Using co-immunoprecipitation assays, we demonstrated that beta- and delta-sarcoglycan interact with alpha-sarcoglycan and these two subunits must be co-expressed for export from the endoplasmic reticulum. Advanced light-microscopic imaging techniques demonstrated that co-expression of beta-sarcoglycan and delta-sarcoglycan is also responsible for delivery to and retention of sarcoglycan subcomplexes at the cell surface. These data suggest that formation of the beta-delta-core may promote the export and deposition of sarcoglycan subcomplexes at the plasma membrane, and therefore identifies a mechanism for sarcoglycan transport.     

Sarcoglycan deficiency in a large Italian population of myopathic patients

Autosomal recessive limb-girdle muscular dystrophies are a heterogeneous group of genetic diseases with a wide spectrum of clinical severity and age of onset; mutations in the gene encoding the dystrophin-associated sarcoglycan proteins (α, β, γ and δ) have recently been shown to cause some cases of these myopathies (primary sarcoglycanopathies, types 2D, 2E, 2C and 2F, respectively). In this study we have examined a large population of Italian myopathic patients to determine the frequency of α-, β- and γ-sarcoglycan deficiency and to correlate molecular defects with clinical phenotypes; to exclude the presence of primary dystrophinopathies both genetic and immunological analysis of dystrophin was performed. We report 12 patients (10 male and 2 female) with deficiency of either one or more sarcoglycan proteins. They were aged 8–56 years with onset between 4 and 30 years of age; they all presented with either mild, moderate or severe limb-girdle involvement associated with elevated blood creatine kinase levels and myopathic pattern at EMG; one was also affected with a mild dilation cardiomyopathy. All patients, except one, showed pathological muscle histological changes. Absence of all three proteins always correlates with severe forms, whereas mild protein deficiencies or isolated partial α-sarcoglycan deficiency correlate with either severe, moderate or mild forms. Received: 31 July 1997 / Revised: 12 December 1997, 23 March 1998 / Accepted: 3 May 1998     

Autosomal recessive muscular dystrophy and mutations of the sarcoglycan complex

Mutations in the genes encoding the dystrophin-associated sarcoglycan proteins (α, β, γ, and δ) (primary sarcoglycanopathies) have recently been shown to cause some cases of the genetically heterogeneous autosomal recessive muscular dystrophies (limb-girdle muscular dystrophy (LGMD) types 2D, 2E, 2C and 2F, respectively). Patients with a primary sarcoglycanopathy are clinically indistinguishable from those with the primary dystrophinopathies. Consequently, a definitive diagnosis can only be achieved through biochemical and molecular analysis. Patient biopsies showing normal dystrophin immunostaining (and/or immunoblot) can be immunostained with antibodies directed against any component of the sarcoglycan complex, and biochemical deficiencies of the sarcoglycan complex can be detected. We have shown, however, that only some of the biochemically-deficient patients are affected with α-, β-, γ- and δ-sarcoglycan mutations. Many will show mutations of an, as yet, unidentified protein. The primary sarcoglycanopathies have been estimated to account for about 5 per cent of muscular dystrophy in patients with normal dystrophin findings.     

Absence of alpha-sarcoglycan and novel missense mutations in the alpha-sarcoglycan gene in a young British girl with muscular dystrophy

An 11-year-old white female presented with progressive proximal muscle weakness and marked calf hypertrophy. Muscle biopsy showed severe dystrophy with normal expression of dystrophin. There was complete absence of the 50kDa dystrophin-associated glycoprotein (alpha-sarcoglycan). DNA analysis showed novel point mutations (one missense and one splicing) in the alpha-sarcoglycan gene at chromosomal location 17q21, confirming the diagnosis of limb-girdle muscular dystrophy type 2D (LGMD-2D). We believe this is one of the first confirmed white cases of primary alpha-sarcoglycanopathy identified in the UK. This case supports the assumption of a wide geographic prevalence of severe childhood onset autosomal recessive muscular dystrophy and genetic heterogeneity. In the future, with improved diagnostic accuracy it is likely that more cases demonstrating primary or secondary deficiency of alpha-sarcoglycan will be identified. We would recommend staining for dystrophin-associated glycoproteins (sarcoglycans) in all new cases of muscular dystrophy with normal dystrophin, and confirmation with DNA analysis where possible.     

Sarcoglycanopathies: can muscle immunoanalysis predict the genotype?

Muscle immunoanalysis of the sarcoglycan complex is an important part of the diagnostic evaluation of muscle biopsies in patients with autosomal recessive limb-girdle muscular dystrophy. Reduced or absent sarcolemmal expression of one or all of the four sarcoglycans (alpha-, beta-, gamma-, delta-sarcoglycan) can be found in patients with limb-girdle muscular dystrophy 2C-F (LGMD2C-F) and also in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). It has previously been suggested that different patterns of sarcoglycan expression could predict the primary genetic defect, and that genetic analysis could be directed by these patterns. In this first UK study we studied 24 genetically characterized patients with sarcoglycan deficient LGMD, in 22 of whom muscle immunoanalysis data were available. Thirteen patients showed alpha-sarcoglycan deficient LGMD2D, 7 patients beta-sarcoglycan deficient LGMD2E, 3 patients gamma-sarcoglycan deficient LGDM2C, and one patient delta-sarcoglycan deficient LGMD2F. Muscle biopsies were analysed in one centre without knowledge of the established genetic diagnosis. Our results demonstrated that residual sarcoglycan expression is highly variable and does not enable an accurate prediction of the genotype. Considering previous reports of sarcoglycanopathy patients with an isolated loss of one sarcoglycan we recommend to use antibodies against all four sarcoglycans for immunoanalysis of skeletal muscle sections. A concomitant reduction of dystrophin and beta-dystroglycan was observed more frequently than previously reported and illustrates the important differential diagnosis of DMD and BMD for sarcoglycan deficient LGMD.     

Becker-like muscular dystrophy in sisters

Two sisters with muscular dystrophy of Becker-like clinical features presented. Muscle weakness was most prominent in the pelvic girdle, but in the elder sister the distal muscles of the lower extremities were also affected. The progression was different in the siblings: The older sister showed a more pronounced deterioration than the younger. The family history was negative in four generations including their brother and youngest sister. Serum creatine kinase activities increased considerably. Electromyogram and muscle biopsy specimens revealed myopathic changes characteristic of muscular dystrophy. Chromosomal analysis confirmed normal 46,XX karyotype. DNA analysis with all cDNA probes spanning the entire dystrophin gene failed to reveal any intragenic deletion or duplication on southern blot. Immunohistochemistry for dystrophin using monoclonal antibodies against the rod and C-terminal domains showed normal continuous staining at the sarcolemma of the muscle fibers in the biopsy specimens of both patients. The results practically exclude the possibility of Xp21 myopathy, and it seems reasonable to classify these patients as having autosomal recessive childhood muscular dystrophy.     

Heart involvement in muscular dystrophies due to sarcoglycan gene mutations

Mutations in the sarcoglycan genes cause autosomal-recessive muscular dystrophies. Because sarcoglycan genes and their protein products are highly expressed both in skeletal and cardiac muscle, patients with these mutations might be expected to be at risk to develop dilated cardiomyopathy. We therefore studied 13 patients with alpha-, beta-, gamma-sarcoglycan gene mutations by thorough cardiological assessment. Electrocardiographic or echocardiographic abnormalities were observed in about 30% of cases showing a severe course of muscular dystrophy. No correlation was found between the presence of cardiac abnormalities and the type of mutation or sarcoglycan gene involved. The cardiac involvement was never severe, but it may be detected in early stages of the muscle disease. The absence of overt cardiac dysfunction may be due to lower sarcoglycan protein expression in cardiac than skeletal muscle or to less sarcolemmal instability at the myocardial level, possibly related to the different distribution of forces generated by contraction of the myocardium with respect to proximal limb-girdle muscles.     

Cardiomyopathic features associated with muscular dystrophy are independent of dystrophin absence in cardiovasculature

The loss of dystrophin results in skeletal muscle degeneration and cardiomyopathy in patients with Duchenne muscular dystrophy. In skeletal muscle, dystrophin strengthens the myofiber membrane by linking the submembranous cytoskeleton and extracellular matrix through its direct interaction with the dystroglycan/sarcoglycan complex. In limb-girdle muscular dystrophy, the loss of the sarcoglycans in cardiovasculature leads to cardiomyopathy. It is unknown whether the absence of dystrophin in cardiomyocytes or cardiovasculature leads to the cardiomyopathy associated with primary dystrophin deficiency. We show here that the cardiomyopathic features of the utrophin/dystrophin-deficient mouse can be prevented by the presence of dystrophin in cardiomyocytes but not in cardiovasculature. Furthermore, restoration of the dystroglycans and sarcoglycans to the cardiomyocyte membrane is not sufficient to prevent cardiomyopathy. These data provide the first evidence that dystrophin plays a mechanical role in cardiomyocytes similar to its role in skeletal muscle. These results indicate that treatment of cardiomyocytes but not cardiovasculature is essential in dystrophinopathies.     

Limb-girdle muscular dystrophy due to emerin gene mutations

BACKGROUND: Emery-Dreifuss muscular dystrophy, caused by EMD gene mutations, is characterized by humeroperoneal muscular dystrophy, joint contractures, and conduction defects and is often associated with sudden cardiac death, even without prior cardiac symptoms. OBJECTIVE: To describe the clinical and molecular features of 2 patients with limb-girdle muscular dystrophy with mutations in EMD. DESIGN: Case reports. SETTING: Academic research. PATIENTS: Two male patients manifested proximal dominant muscle involvement, with minimal or no joint and cardiac involvement. MAIN OUTCOME MEASURES: Muscle biopsy and mutation analysis results. RESULTS: Immunohistochemistry revealed an absence of emerin staining in muscle biopsy specimens. Mutation analysis identified nonsense mutations in EMD. CONCLUSIONS: Mutations in EMD may indicate a limb-girdle muscular dystrophy phenotype. Identification of emerin deficiency among patients with limb-girdle muscular dystrophy is essential to prevent cardiac catastrophe.     

Quadriceps myopathy: forme fruste of Becker muscular dystrophy

We examined dystrophin, the protein product of the Duchenne muscular dystrophy gene, in muscle biopsy specimens from 4 male patients with quadriceps myopathy, all of whom showed a mild and slowly progressive myopathy confined to the quadriceps muscles. All 4 patients had clear abnormalities of dystrophin, and were diagnosed as having Becker muscular dystrophy by both immunofluorescence and immunoblot examinations; that is, dystrophin of an abnormal molecular mass was visualized in muscle cryosections as "patchy" or discontinuous immunostaining at the surface membrane of the muscle fibers. One patient had a brother who showed widespread myopathic changes consistent with typical Becker muscular dystrophy. We conclude that the syndrome called quadriceps myopathy includes a group of forme fruste Becker muscular dystrophy.     

Novel mutations in collagen VI genes: expansion of the Bethlem myopathy phenotype

OBJECTIVE: To investigate the molecular basis of autosomal dominant limb-girdle muscular dystrophy (AD-LGMD) in three large new families. METHODS AND RESULTS: Genome-wide linkage was performed to show that the causative gene in all three families localized to chromosome 21q22.3 (Zmax = 10.3; theta = 0). This region contained the collagen VI alpha1 and alpha2 genes, which have been previously shown to harbor mutations causing a relatively mild congenital myopathy with contractures (Bethlem myopathy). Screening of the collagen VI alpha1 and alpha2 genes revealed novel, causative mutations in each family (COL6A1-K121R, G341D; COL6A2-D620N); two of these mutations were in novel regions of the proteins not previously associated with disease. Collagen VI is a ubiquitously expressed component of connective tissue; however, both limb-girdle muscular dystrophy and Bethlem myopathy patients show symptoms restricted to skeletal muscle. To address the muscle-specific symptoms resulting from collagen VI mutations, the authors studied three patient muscle biopsies at the molecular level (protein expression). A marked reduction of laminin beta1 protein in the myofiber basal lamina in all biopsies was found, although this protein was expressed normally in the neighboring capillary basal laminae. CONCLUSIONS: The authors' studies widen the clinical spectrum of Bethlem myopathy and suggest collagen VI etiology should be investigated in dominant limb-girdle muscular dystrophy. The authors hypothesize that collagen VI mutations lead to muscle-specific defects of the basal lamina, and may explain the muscle-specific symptoms of Bethlem and limb-girdle muscular dystrophy patients with collagen VI mutations.     

Diagnosis of dystrophinopathy by skin biopsy

We studied the expression of dystrophin in skin biopsy samples from 19 patients with neuromuscular diseases. Immunohistochemical procedures for dystrophin analyses were performed using monoclonal antibodies for three different domains. Arrector pili muscles, which are smooth muscles in the skin, expressed dystrophin in the patients with limb-girdle muscular dystrophy (5), facioscapulohumeral muscular dystrophy (1), and spinal muscular atrophy (3), and in normal controls (2). The C-terminus of dystrophin was slightly expressed in the patients with Duchenne muscular dystrophy, whereas the rod domain and N-terminus were absent. In one patient with Becker muscular dystrophy, the expression of dystrophin was reduced. The mosaic of dystrophin positive and negative smooth muscle fibers was observed in a manifesting carrier of Duchenne muscular dystrophy. Our results suggest that skin biopsy is very useful for the diagnosis of Duchenne/Becker muscular dystrophy and manifesting carrier of Duchenne muscular dystrophy, and can be performed even at an advanced stage of the disease.     

Primary adhalin deficiency as a cause of muscular dystrophy in patients with normal dystrophin

In our experience, more than half of muscular dystrophy patients show a primary dystrophinopathy. The underlying cause of muscular dystrophy in the vast majority of patients with normal dystrophin is unknown. Recently, a French family with 4 young siblings showing a muscular dystrophy of unknown progression was shown to have a primary deficiency of ?adhalin,”? the 50-kd dystrophin-associated protein. Here we report the screening of the entire adhalin coding sequence in muscle biopsy specimens from 30 muscular dystrophy patients to (1) determine whether adhalin deficiency is restricted to the French population, (2) determine the incidence of adhalin deficiency in muscular dystrophy patients, and (3) characterize the clinical features and mutations in adhalin-deficient patients. We identified a single African-American girl with childhood-onset muscular dystrophy and adhalin gene mutations. We found her to be a compound heterozygote for two different mutations of the same amino acid (Arg98Cys; Arg98His), one of which was previously identified in the French family. Our results suggest that primary adhalin deficiency in patients with muscular dystrophy but normal dystrophin is relatively infrequent, and that adhalin-deficient patients are not restricted to the French population.     

Muscular dystrophy: advances in research works and therapeutic trials]

Muscular dystrophy is defined as "a group of hereditary disorders with the major symptom of progressive muscle weakness due to muscle fiber degeneration and necrosis". After the discovery of the dystrophin gene and the gene product for Duchenne muscular dystrophy in 1986, there has been remarkable progress in the differential diagnosis and in understanding the pathogenetic mechanism of muscle fiber necrosis. With discoveries of genes responsible for many other disorders, the classification of muscular dystrophy has become more complicated; for instance, there are at least 15 diseases in the limb-girdle muscular dystrophy (LGMD) group, including the autosomal dominant forms, LGMD1A-1E and the recessive forms, LGMD2A-2I. Among them, gene defects in the sarcoglycan complex (sarcoglycanopathy) have been added to LGMD2C-2F. Sarcoglycanopathy seems to be rare in Japan since only 6-7% of LGMD patients had this defect. There are two major possible strategies in treating these patients. One is gene therapy, which is recently being investigated in the mdx mouse by using adenovirus-associated virus (AAV) vector inserted with a microdystrophin gene. Dr Takeda has reported favorable results in mdx mouse muscle with this method. Another is regeneration therapy using stem cells. There are many barriers to overcome to treat patients with stem cells isolated from bone marrow. The most difficult problem to solve is how to culture the stem cells to increase their numbers for application and how to introduce the normal dystrophin gene into these cells.     

Merosin-positive congenital muscular dystrophy: a large inbred family

Large families with congenital muscular dystrophy are rare. We report a clinical, histopathological, immunocytochemical, electrophysiological, radiological and genetic study of 10 cases affected by "pure" CMD belonging to two generations of a large inbred Palestinian family. The disease showed autosomal recessive inheritance. All patients had generalised muscular weakness and hypotonia at birth without arthrogryposis. They had a relatively benign clinical course with stabilisation of the clinical picture at different ages and at variable degrees of severity. The pattern of muscle weakness and wasting was more marked in the proximal upper limb-girdle and trunk muscles. Lower limb muscles were more mildly involved. Serum CK was normal or moderately increased. All patients had normal intelligence, normal computed tomography (CT) scans of the brain and normal somatosensory evoked potentials (SEP). Electromyography (EMG) and muscle biopsy showed morphological changes compatible with muscular dystrophy. Immunocytochemistry for dystrophin, laminin alpha 2 of merosin, and for alpha, beta, gamma sarcoglycans was normal. Linkage analysis excluded all the known loci for CMD, including laminin alpha 2 on chromosome 6q2, the Fukuyama congenital muscular dystrophy locus on 9q3, the integrin alpha 7 locus on chromosome 12q13 and the recently identified locus on 1p35-36. The family we present is clinically and genetically distinct from the already mapped forms of congenital muscular dystrophy. Genetic studies are in progress to localise the gene responsible for this condition.     

Pseudometabolic expression and phenotypic variability of calpain deficiency in two siblings

Two siblings originating from Reunion Island were affected by a limb-girdle muscular dystrophy (LGMD) type 2A and carried the same two mutations in the calpain gene: 946-1 AG→AA, affecting a splice site, and S744G. They demonstrated the clinical variability possible with calpain-3 mutations. Onset was around 20 years of age in each of them. The girl's symptoms mimicked a metabolic myopathy, while her brother, at the same age, presented a classical phenotype of LGMD in an advanced functional stage. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1078–1080, 1998.