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.     

Late-onset autosomal recessive limb-girdle muscular dystrophy with rimmed vacuoles

We report on two siblings with late-onset, limb-girdle muscular dystrophy (LGMD) inherited in an autosomal recessive manner. The LGMD was characterized by many rimmed vacuoles and reduced expression of the laminin beta1 chain in skeletal muscle. Both patients developed a progressive wasting and weakness of limb-girdle muscles in the late forties or early fifties; their facial, ocular, bulbar, and cardiac muscles were not involved. Histopathology of skeletal muscles biopsies showed typical dystrophic changes with many rimmed vacuoles. The immunoreactivity of the laminin beta1 chain was reduced in the muscle fibers, while dystrophin, sarcoglycans, beta-dystroglycan, dysferlin, and other laminin components were normally expressed. A mutation search revealed that no mutation existed in the coding region of the calpain 3, telethonin and UDP-N-acetylglucosamine 2-epimerase/N-acetylmanosamine kinase (GNE) genes. We conclude that this autosomal recessive LGMD is unknown and characterized by its late onset, rimmed vacuoles and reduction of the laminin beta1 chain in muscle fibers.     

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.     

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.     

Autosomal recessive Duchenne-like muscular dystrophy: molecular and histochemical results.

An autosomal recessive disorder which mimics Duchenne muscular dystrophy has long been suspected as a cause of muscular dystrophy in karyotypically normal girls and in both boys and girls with consanguineous parents. Analysis of dystrophin now allows confirmation of the existence of this disorder. We report the results of this analysis in a brother and sister who have the typical clinical features of Duchenne muscular dystrophy, but no demonstrable abnormality in dystrophin or its gene.     

Sarcoglycanopathy]

Sarcoglycanopathy is a group of four autosomal recessive muscular dystrophies whose symptoms are similar to Duchenne muscular dystrophy (DMD). These dystrophies are caused by mutations on anyone of the genes encoding four subunits of sarcoglycan complex which are transmembranous and dystrophin associated proteins. When the protein product of the mutated gene is absent, entire sarcoglycan complex is absent or greatly reduced in amount. This further gives rise to the weak connection between dystrophin and dystroglycan complex. These cause Duchenne-like phenotype. In DMD, dystrophin is absent and sarcoglycan complex is greatly reduced. These similarities in molecular defects in these diseases may cause the similarity in symptoms.     

Severe childhood autosomal recessive muscular dystrophy, mental subnormality and chorea

Severe childhood autosomal recessive muscular dystrophy (SCARMD) is characterized by a severe Duchene muscular dystrophy like phenotype. Most such cases represent alpha or gamma sarcoglycanopathies. Mental subnormality is very uncommon and other central nervous system deficits have not been documented in patients with SCARMD. We report a brother and sister with the SCARMD phenotype, who additionally had static mental subnormality and choreiform movements. Work-up for sarcolgycan genes, dystrophin gene and known causes of mental retardation and chorea was normal.     

Dystrophin and nebulin in the muscular dystrophies

Skeletal muscle from patients with 5 different forms of muscular dystrophy and from 6 fetuses at high risk (95%) for Duchenne muscular dystrophy (DMD) were probed with specific antibodies for the presence of dystrophin and nebulin. Dystrophin was absent in all 5 patients with DMD and 4 of 6 fetuses at high risk for DMD and present in trace amounts in the remaining two. Dystrophin was also undetectable in one borderline DMD/Becker muscular dystrophy (BMD) case and reduced in 2 of 4 cases of BMD. In contrast, dystrophin was present in all 16 biopsies from 4 other types of muscular dystrophy (congenital, limb girdle, Emery-Dreifuss and facioscapulohumeral). Nebulin profiles varied with the type, severity and duration of the dystrophic process. Nebulin was present in 5 of 6 DMD fetal samples but vastly reduced or absent in all samples of clinically manifest DMD.     

CLINICAL AND MOLECULAR PATHOLOGICAL FEATURES OF SEVERE CHILDHOOD AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY IN SAUDI ARABIA

The clinical, biochemical and histochemical features of 14 patients (nine females and five males) with severe childhood autosomal recessive muscular dystrophy (SCARMD) seen at a tertiary hospital in Riyadh from 1982 to 1993 arc described. Onset was at 3 (o 9 (median 3) years and four of five children aged >12 years lost ambulation. Five of the eight pairs of parents were closely consanguineous. The mean creatine kinase was 20 times the upper normal limit. Histochemistry of muscle showed dystrophic features in all cases, and dystrophin was positive in all cases examined (N=6). Three patients (two girls and a boy) were deficient in adhalin, the 50-kDa dystorphin-associated glycoprotein. A boy aged 13 years had rapidly progressing disease. Another boy of the same age (from a family characterized by early onset and slower progression) had normal dystrophin and adhalin. The clinical features conformed with previous observations from Sudan. North Africa and Qatar in the Arabian Peninsula. The disease is common in Saudi Arabia and seems to be more prevalent than Duchenne muscular dystrophy.     

Evaluation of the dystrophin-glycoprotein complex, alpha-actinin, dysferlin and calpain 3 in an autosomal recessive muscular dystrophy in Labrador retrievers

Labrador retrievers suffer from an autosomal recessive muscular dystrophy of unknown aetiology. Dogs affected with this disease develop generalized weakness associated with severe, generalized skeletal muscle atrophy and mild elevations in creatine kinase in the first few months of life. The severity of signs tends to progress over the first year of life but can vary from mild exercise intolerance to non-ambulatory tetraparesis. Beyond 1 year of age, the signs usually stabilize and although muscle mass does not increase, affected dogs' strength may improve slightly. The pathological changes present on muscle biopsy include marked variation in muscle fibre size with hypertrophied and round atrophied fibres present. There is an increased number of fibres with central nuclei and split fibres can be seen. It has been suggested that the disorder is a model for limb-girdle muscular dystrophy. In recent years, mutations in genes encoding the proteolytic enzyme, calpain 3, a novel protein named dysferlin, and components of the dystrophin-glycoprotein complex have been identified as causes of autosomal recessive limb-girdle muscular dystrophy. We have evaluated these proteins in normal dogs and in three Labrador retrievers with autosomal recessive muscular dystrophy using immunohistochemistry and Western blot analysis on frozen skeletal muscle. The results demonstrate that dystrophin, the sarcoglycans, alpha-actinin, dysferlin and calpain 3 are present in the normal and affected dogs. We conclude that this autosomal recessive muscular dystrophy is not due to a deficiency of alpha-actinin, or any of the known autosomal recessive limb-girdle muscular dystrophy proteins, although we cannot rule out a malfunction of any of these proteins.     

On symptomatic heterozygous alpha-sarcoglycan gene mutation carriers

Mutations in the human alpha-sarcoglycan gene on chromosome 17q21.2 have been shown to cause a severe childhood autosomal recessive muscular dystrophy, a less severe limb girdle muscular dystrophy, exercise intolerance, or asymptomatic hyperCKemia. Here, we describe the clinical findings in a German family harboring a 371 T > C (Ile124Thr) missense mutation in the alpha-sarcoglycan gene. Whereas our index patient, an 11-year-old girl homozygous for this mutation, presented with a severe Duchenne-like phenotype, 7 out of 12 heterozygous mutation carriers from three generations showed mild to moderate scapular winging. In analogy to symptomatic female dystrophinopathy carriers, our results suggest that heterozygous alpha-sarcoglycan gene mutation carriers can be symptomatic with selective muscle weakness. This finding may be attributed to an additional negative variation in a yet unknown modifier gene essential to the function of the sarcoglycan complex in shoulder girdle muscles.     

Extraocular muscle is spared despite the absence of an intact sarcoglycan complex in gamma- or delta-sarcoglycan-deficient mice

Models of the dystrophin-glycoprotein complex do not reconcile the novel sparing of extraocular muscle in muscular dystrophy. Extraocular muscle sparing in Duchenne muscular dystrophy implies the existence of adaptive properties in these muscles that may extend protection to other neuromuscular diseases. We studied the extraocular muscle morphology and dystrophin-glycoprotein complex organization in murine targeted deletion of the gamma-sarcoglycan (gsg(-/-)) and delta-sarcoglycan (dsg(-/-)) genes, two models of autosomal recessive limb girdle muscular dystrophy. In contrast to limb and diaphragm, the principal extraocular muscles were intact in gsg(-/-) and dsg(-/-) mice. However, central nucleated, presumptive regenerative, fibers were seen in the accessory extraocular muscles (retractor bulbi, levator palpebrae superioris) of both strains. Skeletal muscles of gsg(-/-) mice exhibited in vivo Evans Blue dye permeability, while the principal extraocular muscles did not. Disruption of gamma-sarcoglycan produced secondary displacement of alpha- and beta-sarcoglycans in the extraocular muscles. The intensity of immunofluorescence for dystrophin and alpha- and beta-dystroglycan also appeared to be slightly reduced. Utrophin localization was unchanged. The finding that sarcoglycan disruption was insufficient to elicit alterations in extraocular muscle suggests that loss of mechanical stability and increased sarcolemmal permeability are not inevitable consequences of mutations that disrupt the dystrophin-glycoprotein complex organization and must be accounted for in models of muscular dystrophy.     

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     

Dystrophin and beta-dystroglycan in photoreceptor terminals from normal and mdx3Cv mouse retinae.

Mutations in the dystrophin gene cause muscular dystrophy as well as cognitive impairments, including an abnormal dark-adapted electroretinogram. To investigate the basis for the ocular phenotype, we analysed dystrophin and the dystrophin-associated protein beta-dystroglycan in retinae from mdx3Cv mice. This strain has a mutation in the dystrophin gene and abnormalities in the electroretinogram which are similar to those of muscular dystrophy patients. Despite an overall reduction of all dystrophin isoforms and of beta-dystroglycan in retinal tissue from mutant mice, we observed no apparent change in the histotypic layering of the retina, or in the ultrastructure of several specific cell types, including rods and cones. In retinae from wild type and mdx3Cv mice, dystrophin and beta-dystroglycan were concentrated in small extensions of rod and cone photoreceptor terminals protruding into the outer plexiform layer. Beta-dystroglycan but not dystrophin was also clustered around the inner limiting membrane and the capillary basal laminae. While the labelling pattern around the basal laminae was not altered in the mutant mice, we found that the area as well as the intensity of the dystrophin and beta-dystroglycan immunoreactivity associated with the terminals of rod photoreceptors were severely reduced. The same parameters were much less affected in cone terminals. These results show, that dystrophin and beta-dystroglycan are differentially distributed in the retina, and that a severe reduction of dystrophin has no gross effect on retinal structure, but could influence intraretinal signalling at the level of the photoreceptor terminals. Moreover, the mutation in mdx3Cv mice has a selective effect on rods, providing an explanation for the altered electroretinogram.     

Early onset autosomal dominant progressive muscular dystrophy presenting in childhood as a Becker phenotype--the importance of dystrophin and molecular genetic analysis.

We present two cases of autosomal dominant limb girdle muscular dystrophy in a father and son. Both presented in childhood with a classical Becker muscular dystrophy phenotype. The father had initially been informed that he would not have affected children. After the diagnosis of muscular dystrophy in the son, immunoblot analysis was performed on muscle and revealed normal dystrophin. The polymerase chain reaction did not show any deletions in the dystrophin gene, and the father's dystrophin gene was not passed to his son. These cases demonstrate that autosomal dominant muscular dystrophy may present in childhood, and that dystrophin and molecular genetic analyses should be performed when considering the diagnosis of childhood muscular dystrophy, even in the presence of a classical phenotype.     

Variable histological expression of dystrophinopathy in two females

We report two carriers of Xp21 muscular dystrophy with unusual clinical manifestations and striking variability of dystrophin deficiency within the same muscle biopsy. The first patient was a 60-year-old nun with recent onset of cramps and proximal weakness, mimicking an acquired myopathy. Muscle biopsy disclosed slight alterations in one sample and severe dystrophic changes in another; dystrophin was absent in 7% fibers in the former specimen and in 60% in the second. X inactivation was skewed with 90% cells inactivating the same X chromosome. The second patient was a 17-year-old girl with hyperCKemia, learning disability and a family history of X-linked muscular dystrophy. Muscle biopsy displayed slight fiber size variability and some internal nuclei; dystrophin was absent only in one muscle fiber. A second sample with the same morphological features demonstrated dystrophin deficiency with mosaic distribution. The pattern of X inactivation was normal. These cases emphasize the variability of histopathological changes and dystrophin deficiency in Xp21 muscular dystrophy carriers and the risk of sampling errors in muscle biopsy. Received: 26 August 1998 / Revised, accepted: 30 November 1998     

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.     

Limb-girdle syndrome: a genetic study of 22 large Brazilian families. Comparison with X-linked Duchenne and Becker dystrophies

The differential clinical diagnosis between the X-linked muscular dystrophies (DMD and BMD) and autosomal recessive limb-girdle muscular dystrophy (LGMD), which is extremely important for genetic counseling, may be very difficult. The aim of the present report is to describe clinical and laboratory findings in patients from large families, with AR inheritance, in an attempt to characterize better cases which have been diagnosed as LGMD compared with the X-linked forms. The main features analysed are: age of onset and of confinement to a wheelchair, reproductive performance, serum enzymes (CK and PK) and dystrophin assessment (through immunohistochemistry and Western blot). Twenty-two families, with 62 affected patients diagnosed as limb-girdle muscular dystrophy, were included in this report. In 19 families, the patients had a milder clinical course, while in the remaining 3, the progression of the disease was continuous and clinically similar to X-linked DMD ("DMD-like"). A high consanguinity rate was observed among the parents of the affected patients (77%). No major clinical difference was observed between the X-linked and the AR forms. However, muscle dystrophin was found qualitatively and quantitatively normal in the autosomal forms but absent or abnormal in the X-linked ones. The reproductive performance was significantly higher for male than female patients. In addition, a surprising finding was the significantly greater fitness estimated for male LGMD cases as compared with Becker patients of comparable age studied in our center. The implications of such findings are discussed.     

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.     

Duchenne muscular dystrophy in Tunisia: a clinical and morphological study of 77 cases.

Two types of progressive muscular dystrophy occur in Tunisian children. The first type is characterized by normal dystrophin assays and affects girls and boys in an autosomal recessive pattern of inheritance. The second type has the features of the typical Duchenne muscular dystrophy (DMD) and has abnormal dystrophin. Between 1974 and 1986, 77 patients with Duchenne muscular dystrophy were examined, 66 were biopsied. Among affected siblings and within family kindreds, we observed both clinical and histopathological variability. However, there was a close correlation between the clinical condition and the biopsy findings in each case, allowing accurate prediction of the patient's course and probable duration of the disease.