Late onset in dysferlinopathy widens the clinical spectrum.

LGMD2B, Miyoshi Myopathy and Distal Anterior Compartment Myopathy are caused by mutations in the dysferlin gene (DYSF) leading to progressive muscular weakness and wasting with onset usually within the second or third decade of life. We here present a patient with disease onset at 73 years. The presenting symptom was exercise-induced stiffness of the trunk and proximal leg muscles without major progression over a period of 12 years. Gastrocnemius muscle biopsy revealed dystrophic morphology and biochemical depletion of dysferlin, while sequence analysis revealed compound heterozygous splicing mutations of the dysferlin gene. This case represents the eldest age of onset of dysferlinopathy reported so far and widens the clinical spectrum of this disease.

Diagnostic protein expression in human muscle biopsies.

Using immunohistochemistry in diagnosing neuromuscular diseases is meant to enhance the diagnostic yield in two ways. The first application aims at visualizing molecules which are developmentally, neurally, and/or immunologically regulated and not expressed by normal muscle. They are upregulated in pathological conditions and may help assign a given muscular biopsy to one of the main diagnostic entities (muscular dystrophies, inflammatory myopathy, neurogenic atrophy). In the past, muscle-specific molecules with a defined expression pattern during fetal myogenesis served as antigens, with the rationale that the developmental program was switched on in new fibers. Recently, myofibers in diseased muscle are thought of as targets of stimuli which are released by macrophages in muscular dystrophy, by lymphocytes in inflammatory myopathies, or by a lesioned peripheral nerve in neurogenic atrophies. This has somewhat blurred the borders between the diagnostic groups, for certain molecules, e.g. cytokines, may be upregulated after experimental necrotization, denervation, and also in inflammatory myopathies. In the second part of this review we summarise the experiences of a Centre in the North of England that specialises in the diagnosis and clinical support of patients with muscular dystrophy. Emphasis is placed on the use of protein expression to guide mutation analysis, particularly in the limb-girdle muscular dystrophies (a group of diseases that are very difficult to differentiate on clinical grounds alone). We confirm that genetic analysis is essential to corroborate the results of protein analysis in certain conditions (particularly in calpainopathy). However, we conclude that analysing biopsies for abnormal protein expression is very useful in aiding the decision between alternative diagnoses.

Dystrophin nonsense mutation induces different levels of exon 29 skipping and leads to variable phenotypes within one BMD family.

Within one X-linked muscular dystrophy family, different phenotypes for three males occurred: (1) a severely affected Becker patient with cardiomyopathy, (2) a mildly affected Becker patient, and (3) an apparently healthy male with elevated serum CK levels. In the muscle biopsy specimen of patient2 one out of four antibodies (NCL-DYS1) showed absence of dystrophin. The protein truncation test detected a truncated dystrophin for both muscle tissue and lymphocytes of this patient next to an additional near normal size fragment in muscle. Genomic sequence analysis revealed a nonsense mutation in exon 29 (4148C > T) of the dystrophin gene. Sequence analysis of the mRNA fragment of the larger peptide showed skipping of exon 29, restoring an open reading frame. Consequently, the epitope of the antibody NCL-DYS1 is mapped to exon 29. The variable clinical features of the three relatives from healthy to severely affected therefore seems to be related to the level of skipping of exon 29. This finding underscores the future potential of gene therapeutic strategies aimed at inducing exon skipping in Duchenne muscular dystrophy, to generate a much milder disease.

Becker muscular dystrophy with onset after 60 years.

We report an unusual example of Becker muscular dystrophy in which the patient was asymptomatic until his mid-60s, and diagnosis was not made until he was 67 years old. The patient had the unusual deletion of exons 3 to 9 within an actin-binding region of the dystrophin gene, removal or disruption of which is almost invariably associated with a more severe clinical phenotype. Our case illustrates the danger of drawing conclusions about possibly symptomatic deletions without lifelong follow-up.

Optimized protein diagnosis in the autosomal recessive limb-girdle muscular dystrophies.

The genes for six forms of recessive muscular dystrophy have so far been identified, although more are certain to be revealed. Differential diagnosis on clinical grounds alone can be very difficult, so a classification system based on the underlying molecular defect has been introduced. Muscle biopsies are taken for routine diagnostic histopathology, and the various proteins implicated in muscular dystrophy can be analysed immunologically and the results used to indicate where to start searching for gene mutations. A flow diagram is presented which demonstrates how such protein analysis could be optimized.

Immunomarkers for molecular mass.

The proteins known to cause different forms of muscular dystrophy cover a wide range of sizes, and it is now possible to use a cocktail of antibodies to generate a ladder of molecular mass markers on Western blots of human skeletal muscle.

Dysferlinopathy (LGMD2B): a 23-year follow-up study of 10 patients homozygous for the same frameshifting dysferlin mutations.

The limb-girdle muscular dystrophies are a group of inherited neuromuscular disorders which are clinically and genetically heterogeneous. We have been able to carry out a follow-up study on 10 patients from a large Palestinian family with a confirmed mutation in the dysferlin gene. These patients have been followed for more than 23 years since the onset of the disease. They all had normal developmental milestones. The onset of the disease was usually in the second decade, more rarely in the third and fourth decades. The first symptoms were difficulty with running and climbing stairs. Patients showed a distinct type of gait due to the unique pattern of muscle involvement which was characterised by early involvement of the posterior muscle compartment of the thighs and legs (hamstrings, adductors, gastrocnemius and soleus). The shoulder and upper limb musculature became involved later, especially supra and infraspinatus and biceps. In the early stages of disease these patients may clinically show only proximal lower limb-girdle muscle weakness; however, the use of muscle imaging techniques were very important, always detecting in these patients also distal lower limb muscle involvement, so that the pattern of muscle involvement found in dysferlin deficiency may not strictly conform to the definition of limb-girdle muscular dystrophy. The pattern of muscular dystrophy is essentially uniform and has clearly distinct features (involving mainly the initial pattern of muscle involvement and the mode of gait) which differ significantly from the well reported clinical features associated with sarcoglycanopathy, calpainopathy and Miyoshi myopathy.

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.

Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice.

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.

The phenotype of calpainopathy: diagnosis based on a multidisciplinary approach.

Calpainopathy (LGMD2A) is the most common type of autosomal recessive limb-girdle muscular dystrophy. We performed a systematic clinical evaluation in 13 calpainopathy patients from 11 families, with particular attention to the pattern of muscle involvement. Eleven patients had a muscle biopsy with deficiency of calpain 3 on western blotting. The other two patients were not biopsied as they were siblings from the same families. Confirmatory CAPN3 mutations were detected in seven patients. The age at presentation was 2-45 years, wider than previously reported. We confirm the highly characteristic and recognisable phenotype of predominant muscular atrophy with early pelvic girdle involvement, relative sparing of the hip abductors, scapular winging and abdominal laxity. Early primary contractures were also a prominent feature in this group, expanding the breadth of the phenotype. Recognition of the clinical pattern of calpainopathy is of diagnostic significance. It is important, especially in sporadic cases, in targeting and interpreting laboratory investigations in order to provide accurate diagnostic and prognostic information.

Calpain3 expression during human cardiogenesis.

Transcripts of calpain3, the gene involved in limb girdle muscular dystrophy type 2A, appear in organs other than the skeletal muscle during human development, the first of which being the early embryonic heart. We examined more precisely the spatio-temporal transcription pattern of calpain3 during human cardiogenesis and the appearance of its protein in fetal tissues, and correlated it to titin expression. Different events of the heart's maturation can be recognized: (i) the presence of titin RNA or protein constitute very precocious developmental cardiac markers appearing before the fusion of the two lateral endocardial tubes; (ii) the disappearance of calpain3 RNA from the ventricular compartment later in the embryonic heart. Finally, although calpain3 transcripts are present in the heart, the corresponding protein is not detected elsewhere than in skeletal muscle.

Abnormalities in alpha-, beta- and gamma-sarcoglycan in patients with limb-girdle muscular dystrophy.

We have identified 12 cases from a group of 45 patients with early onset limb-girdle muscular dystrophy (LGMD), who have a deficiency of the 50 kDa dystrophin-associated glycoprotein, alpha-sarcoglycan. An additional male sibling of one case was also studied clinically. All 12 patients showed a concomitant, but variable, deficiency of alpha-, beta- and gamma-sarcoglycan. None of our patients had a defect in only one component of the sarcoglycan complex. Molecular analysis confirmed that a total absence of one sarcoglycan, associated with reduced expression of the other two, indicates a primary defect. Immunocytochemistry is thus useful for directing molecular studies. Morphological features not usually observed in Xp21 dystrophies were peripheral accumulations of mitochondria, discrete core-like areas, and nemaline rods in one case. Clinical severity and progression was variable between and within families but early loss of ambulation, at or before the age of 12 years, was associated with a total absence of gamma-sarcoglycan. Common clinical features were calf hypertrophy, contractures of the tendo achilles, lumbar lordosis, winging of the scapulae, weak hamstrings and weak neck muscles. All cases had grossly elevated serum creatine kinase. In contrast to patients with Duchenne muscular dystrophy (DMD), our patients with sarcoglycan deficiencies had normal early motor milestones, normal intellect, and good respiratory and cardiac function. Our data confirm that the sarcoglycan complex acts as a unit and that morphological and clinical features can distinguish patients with defects in the sarcoglycans from those with Xp21 dystrophy. In our group of patients prognosis is better than in DMD, but clinical variability makes this difficult to predict in isolated cases.

Muscle membrane-skeleton protein changes and histopathological characterization of muscle-eye-brain disease.

Muscle-eye-brain disease belongs to congenital muscular dystrophies with central nervous system abnormalities. The etiology of MEB is still unknown, but abnormal immunoreactivity for laminin-2 has been reported. To evaluate disease progression in muscle tissue, 32 biopsy specimens from 17 muscle-eye-brain patients were analysed. The samples of four patients were studied by immunohistochemical techniques and by quantitative Western blotting. The samples showed a great variation in the muscle pathology. Regenerative fibers and mild fiber size variation were present in over 60%. At infancy, necrotic and regenerative fibers were common, while fat infiltration was the most prominent finding in the age group over five years. In quantitative studies, the amount of laminin alpha 2 chain was clearly reduced to 10-20% of normal. In contrast, laminin beta 2 chain was overexpressed in the Western blotting studies. These findings may reflect a yet unidentified primary disturbance in the basement membrane composition and function.

Strategy for mutation analysis in the autosomal recessive limb-girdle muscular dystrophies.

We describe a strategy for molecular diagnosis in the autosomal recessive limb-girdle muscular dystrophies, a highly heterogeneous group of inherited muscle-wasting diseases. Genetic mutation analysis is directed by immunoanalysis of muscle biopsies using antibodies against a panel of muscular dystrophy-associated proteins. Performing the molecular analysis in this way greatly increases the chance that mutations will be found in the first gene examined. The use of this strategy can significantly decrease the time involved in determining the genetic fault in a patient with a clinical diagnosis of recessive limb-girdle muscular dystrophy, as well as having a feedback effect, which is useful in helping clinicians to identify subtle clinical differences between the subtypes of the disease. The use of this approach has so far helped us to identify mutations in ten sarcoglycanopathy (limb-girdle muscular dystrophy 2C-2F) patients, and seven calpainopathy (limb-girdle muscular dystrophy 2A) patients.

Molecular analysis of a spontaneous dystrophin 'knockout' dog.

We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.

Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies).

Dysferlin is the protein product of the gene (DYSF) that is defective in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy. Calpain 3 is the muscle-specific member of the calcium activated neutral protease family and primary mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A. The functions of both proteins remain speculative. Here we report a secondary reduction in calpain 3 expression in eight out of 16 patients with a primary dysferlinopathy and clinical features characteristic of limb girdle muscular dystrophy type 2B or Miyoshi myopathy. Previously CAPN3 analysis had been undertaken in three of these patients and two showed seemingly innocuous missense mutations, changing calpain 3 amino acids to those present in the sequences of calpains 1 and 2. These results suggest that there may be an association between dysferlin and calpain 3, and further analysis of both genes may elucidate a novel functional interaction. In addition, an association was found between prominent expression of smaller forms of the 80 kDa fragment of laminin alpha 2 chain (merosin) and dysferlin-deficiency.

Dystrophin and the dystrophin-associated glycoprotein, beta-dystroglycan, co-localize in photoreceptor synaptic complexes of the human retina.

Mutations in the gene encoding for dystrophin, a membrane-associated cytoskeletal protein of muscle and several non-muscle cells, are the cause of Duchenne muscular dystrophy and Becker muscular dystrophy. Patients suffering from Duchenne muscular dystrophy have recently been shown to display an abnormal b-wave of the electroretinogram, suggesting that dystrophin is important for normal retinal transmission. In the retina, dystrophin has been localized in the outer plexiform layer where dystrophin co-localizes with postsynaptic markers of photoreceptor synaptic complexes. In the present study we addressed the question of whether two major dystrophin-associated integral membrane proteins of the muscular plasma membrane, beta-dystroglycan and adhalin, are also present in photoreceptor synaptic complexes. By double immunostaining and immunoblotting we show here that beta-dystroglycan is expressed in the human retina where it co-localizes with dystrophin in photoreceptor synaptic complexes most likely on the postsynaptic side. Adhalin was not detected in the retina. Since beta-dystroglycan is a member of a transmembrane supramolecular complex thought to be important for differentiation of the neuromuscular junction, it is an attractive hypothesis that dystroglycan (linked to dystrophin) might also play a similar role in differentiation of the photoreceptor synapse. A further outcome of this study is that beta-dystroglycan is not only present in the neuromuscular junction but also associated with a well-defined synaptic complex of the central nervous system. These findings indicate a more general role of this dystrophin-associated membrane protein in synaptic functions.

Dysferlin protein analysis in limb-girdle muscular dystrophies.

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD21 (19q13.3). We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency. In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B. Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients. Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle. Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin. Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (approximately 14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.