A mutation in the dystrophin gene selectively affecting dystrophin expression in the heart.

We have previously shown in a large X-linked pedigree that a deletion removing the dystrophin muscle promoter, the first muscle exon and part of intron 1 caused a severe dilated cardiomyopathy with no associated muscle weakness. Dystrophin expression was present in the muscle of affected males and transcription studies indicated that this dystrophin originated from the brain and Purkinje cell isoforms, upregulated in this skeletal muscle. We have now studied dystrophin transcription and expression in the heart of one member of this family. In contrast to the skeletal muscle, dystrophin transcription and expression were absent in the heart, with the exception of the distal Dp71 dystrophin isoform, normally present in the heart. The 43- and 50-kD dystrophin-associated proteins were severely reduced in the heart, despite the presence of Dp71, but not in skeletal muscle. The absence of dystrophin and the down-regulation of the dystrophin-associated proteins in the heart accounted for the severe cardiomyopathy in this family. The mutation present in these males selectively affects dystrophin expression in the heart; this could be secondary to the removal of cardiac-specific regulatory sequences. This family may represent the first example of a mutation specifically affecting the cardiac expression of a gene, present physiologically in both the skeletal and cardiac muscles.

Calmodulin-binding profiles for nebulin and dystrophin in human skeletal muscle.

Nebulin and dystrophin are two high-molecular-mass skeletal muscle proteins that have both been associated with the defective gene in Duchenne muscular dystrophy, although the function of neither protein is known. Other high-molecular-mass, calmodulin-binding proteins have recently been implicated in regulating calcium release from skeletal muscle. Western blots of human skeletal muscle biopsy samples were probed with biotinylated calmodulin; nebulin was identified as a prominent high-molecular-mass calmodulin-binding protein but dystrophin did not bind detectable amounts of biotinylated calmodulin. Dystrophin was absent in a Duchenne muscle biopsy.

The role of Coxsackie B viruses in the pathogenesis of myocarditis, dilated cardiomyopathy and inflammatory muscle disease.

Coxsackie B viruses are members of the family Picornaviridae which have been associated by retrospective serology with a range of muscle diseases, particularly myocarditis, dilated cardiomyopathy and epidemic pleurodynia (epidemic myalgia or Bornholm disease). It has been proposed that virus-induced myocarditis disposes to the development of idiopathic dilated cardiomyopathy. However, despite many attempts, isolation of infectious virus or immunofluorescent detection of virus-specific antigens in the affected tissue is rare, although virus may be found in faeces early in infection. This discrepancy awaited the development of nucleic acid probes to resolve the problem of whether virus was present consistently in myocardium or other muscle tissues. We report here the synthesis of Coxsackie B virus-specific complementary DNA (cDNA) probes and their use in molecular hybridizations to quantitative slot-blots of RNA prepared from either endomyocardial or skeletal muscle biopsy specimens. Of 50 patients with histologically proven myocarditis or dilated cardiomyopathy, 28 (56%) had an endomyocardial biopsy specimen positive for the presence of Coxsackie B virus-specific RNA. Twenty-two patients with other cardiac diseases of known aetiology, unrelated to virus infection, were all negative. Multiple biopsies were obtained from 20 patients with myocarditis or dilated cardiomyopathy and 15 of these (75%) had at least one biopsy specimen positive, indicating the focal nature of the disease. In analogous investigations, Coxsackie B virus-specific RNA was detected in four out of seven single skeletal muscle biopsy specimens from patients suffering from juvenile dermatomyositis, and one out of two patients with adult polymyositis. Ten muscle controls, either normal or Duchenne muscular dystrophy, were negative for virus RNA.

Early onset, autosomal recessive muscular dystrophy with Emery-Dreifuss phenotype and normal emerin expression.

OBJECTIVE: To describe the clinical and histopathologic picture of a childhood-onset, severe variant of scapuloperoneal MD with rigidity of the spine. BACKGROUND: Rigidity of the spine is a feature of numerous syndromes, including X-linked Emery-Dreifuss MD, Bethlem myopathy, and the rigid spine syndrome. These are, however, relatively static or very slowly progressive neuromuscular disorders, usually associated with preserved ambulation into adult life. PATIENTS AND METHODS: Five unrelated children (three boys and two girls) presented in the first 2 years of life with poor neck control, waddling gait, and frequent falls. Early wasting of the distal leg muscles, biceps, triceps, and neck muscles was noted in all patients, and all had contractures and severe rigidity of the spine. The condition progressed rapidly, and all patients lost ambulation before the age of 8 years. Cardiac function was normal in all. RESULTS: Creatine kinase was moderately elevated in all, and muscle biopsy specimens showed nonspecific dystrophic changes with normal expression of dystrophin, the sarcoglycans, and laminin alpha2, alpha5, beta1, and gamma1 chains. Emerin expression was normal in two of the boys whose tissue was available for study. CONCLUSIONS: The distribution of weakness, wasting, and contractures of the patients described resembled Emery-Dreifuss MD, but the rapid progression of weakness and contractures and the involvement of both sexes together with normal emerin expression suggest that this form is not X-linked Emery-Dreifuss MD. We suggest that these patients represent a severe MD characterized by early onset distal wasting and severe rigidity of the spine, with probable autosomal recessive inheritance.

An X-linked disease of the nervous system with disordered copper metabolism and features differing from Menkes disease.

We studied 2 of 4 affected boys with a new disease associated with abnormalities of copper metabolism. The four cases occurred in two generations of a family. This syndrome was similar to Menkes disease in some respects: X-linked recessive inheritance, marked psychomotor retardation with seizures, low serum copper and ceruloplasmin levels, and a block in gut copper absorption. There were also striking differences from Menkes disease. Patients had normal birthweight at term, no hypothermia, and survived beyond the usual Menkes age group with static neurologic disease including hypotonia and choreoathetosis. In addition, general examination of both children was unremarkable apart from undescended testes and growth retardation. The hair, facies, and skin were normal and there was no radiologic evidence of bony changes. Detailed studies of copper absorption were performed.

Phenotypic heterogeneity of spinal muscular atrophy mapping to chromosome 5q11.2-13.3 (SMA 5q).

We made phenotypic analysis of 14 families with spinal muscular atrophy (SMA) linking to chromosome 5q11.2-13.3 (SMA 5q), and 2 that may not map to this locus, to assess clinical symptoms among SMA families known to result from mutation at the identical gene/locus. Although the current number of families is still small, the correlation of clinical phenotype and molecular genotype supports 2 observations. First, SMA mutations at the 5q locus present with a broad continuum of clinical abnormalities, and 2nd, the single clearly unlinked family presents with an unusual phenotype characterized by relatively late onset and early death. Thus, there are as yet no unambiguous cases of typical SMA families that are clearly unlinked to the locus at 5q-ie, no clear cases of nonallelic heterogeneity. Analysis of SMA 5q families supports the view that, with certain exceptions, there is little phenotypic intrafamilial variability. When families were ranked by severity of disease there was a strong correlation with age of onset. Onset within the 1st few months was associated with early death, but not in all cases. With rare exception, onset after 1 year of age was associated with less severe disease and greater longevity.

Autosomal recessive inheritance of RYR1 mutations in a congenital myopathy with cores.

Central core disease (CCD) is a congenital myopathy due to dominant mutations in the skeletal muscle ryanodine receptor gene (RYR1). The authors report three patients from two consanguineous families with symptoms of a congenital myopathy, cores on muscle biopsy, and confirmed linkage to the RYR1 locus. Molecular genetic studies in one family identified a V4849I homozygous missense mutation in the RYR1 gene. This report suggests a congenital myopathy associated with recessive RYR1 mutations.

Fatal rhabdomyolysis complicating general anaesthesia in a child with Becker muscular dystrophy.

A 6-yr-old boy who presented with brown urine due to myoglobinuria and who was otherwise virtually asymptomatic was diagnosed as having Becker muscular dystrophy on the basis of a greatly elevated creatine kinase, muscle biopsy, dystrophin analysis, and a deletion of exons 3-7 in the dystrophin gene. Fifteen months later, during a general anaesthetic for dental treatment, he had a cardiac arrest associated with acute rhabdomyolysis, hyperkalaemia and hypocalcaemia. He died 4 days later. This case is reported to highlight this rare but potentially fatal complication of anaesthesia in muscular dystrophy, and to discuss possible ways of preventing such a catastrophe.

Steroids in Duchenne muscular dystrophy; pilot study of a new low-dosage schedule.

Prednisolone has been shown to improve strength in Duchenne dystrophy, the improvement starting within 10 days of treatment and reaching a maximum by 3 months, and then plateauing. Unfortunately this has been associated with an unacceptably high level of side effects. In an attempt to obtain the benefit of steroids without the side effects we devised an intermittent low dosage schedule, with 0.75 mg kg-1 day-1 for 10 days at the beginning of each calendar month. To date 32 boys with Duchenne dystrophy have been enrolled into an open randomized trial. Preliminary data show an influence on strength at 6 months but a slow decline at 12 and 18 months. The weight gain and other side effects have been much less than with continuous therapy.

The natural history of type I (severe) spinal muscular atrophy.

The clinical features of 36 patients who satisfied the diagnostic criteria for type I (severe) spinal muscular atrophy (Werdnig-Hoffmann disease) are reported. Survival data for both the whole cohort and for groups within the cohort subdivided on the age of onset are presented. These data suggest that the patients with onset at birth or within the first 2 months of life have a more uniformly poor prognosis with earlier death. This is of potential importance in any therapeutic trials in the future whose outcome may be based on length of survival.

Cardiac transplantation in Becker muscular dystrophy.

Becker muscular dystrophy is associated with abnormal cardiac features in about 75% of cases; up to one-third will develop ventricular dilatation leading to congestive cardiac failure. As this form of muscular dystrophy is relatively benign, failure to respond to medical treatment warrants assessment for cardiac transplantation.

Clinical phenotype in congenital muscular dystrophy: correlation with expression of merosin in skeletal muscle.

It has recently been shown that merosin, an extracellular matrix protein linked to the dystrophin-associated glycoproteins, is deficient in a proportion of patients with classical congenital muscular dystrophy (CMD). We have undertaken a detailed study of the clinical features and brain imaging in 24 cases of CMD in relation to the merosin status. Immunocytochemistry showed that merosin was present in 13 cases and markedly deficient in 11. In the merosin-positive cases, the maximum motor achievement was independent walking in 11, walking with support in one and sitting unsupported in one (currently 18 months old). In contrast, none of the merosin-deficient cases achieved independent ambulation. Two achieved walking with support, nine standing with support. In addition, nine of the 11 merosin-deficient cases had a creatine kinase level greater than 2000 whereas only one merosin-positive case had this degree of elevation. Magnetic resonance imaging of the brain was carried out on 15 of the children. All eight merosin-positive cases had normal scans whereas all seven of the merosin-deficient cases had significant changes in the white matter. This study has demonstrated that children with merosin-deficient CMD have a more severe clinical phenotype and associated white matter changes on brain imaging.

Congenital symmetrical weakness of the upper limbs resembling brachial plexus palsy: a possible sequel of drug toxicity in first trimester of pregnancy?

We report a 14-month-old girl with a symmetrical paralysis from birth, limited to the upper limbs and resembling a severe, complete bilateral brachial plexus palsy. The presence of dimples over the wrists, shoulders and scapulae and abnormal palmar dermatoglyphics suggested an early prenatal onset. Previous reports and the course of the disease in our case suggest this sporadic condition is not progressive. Although no definitive causative factor has been identified in previously reported cases, the affection in our case is possibly related to Debendox (Bendectin) and nitrofurantoin taken in early pregnancy for nausea and renal tract infection, respectively.

Familial concordance of brain magnetic resonance imaging changes in congenital muscular dystrophy.

Cerebral white matter changes have been described in a significant number of individual patients with "pure" congenital muscular dystrophy without clinical evidence of central nervous system involvement. The cause for the imaging changes is unknown but it is possible that they are the result of abnormal expression in the brain of the gene also responsible for the muscular dystrophy. In this study magnetic resonance imaging of the brain was performed on seven sibling pairs with congenital muscular dystrophy and normal intelligence to establish whether imaging changes are consistent within families. Diagnosis of congenital muscular dystrophy was based on clinical and muscle biopsy findings. Children from two families had normal scans; the remaining five sibling pairs showed white matter changes and within each family the changes were virtually identical in severity and distribution. Our data indicate that the central nervous system changes are consistent within individual families, suggesting that they probably relate to the mutation in the congenital muscular dystrophy genes involved in the respective families.

No evidence of association between apolipoprotein E genotype and phenotypic severity in childhood onset proximal spinal muscular atrophy.

The survival motor neuron (SMN) gene is present in two copies on chromosome 5q13 and the evidence is now compelling that mutations in the telomeric copy (SMNt) of the gene underlie childhood onset proximal spinal muscular atrophy (SMA). There is a correlation between the number of centromeric SMN gene copies (SMNc) and the clinical severity of the disease but this relationship is not absolute. Allelic variants of the apolipoprotein E (APOE) gene encoded on chromosome 19q are known to influence the prognosis and risk in a number of neurological disorders. We have therefore genotyped 166 unrelated cases of SMA to determine whether the presence of specific APOE genotypes correlates with severity of disease. The study failed to show the influence of any particular APOE genotype on disease severity, with specifically APOE epsilon4 being no more common in the milder SMA forms and APOE epsilon2 not over represented in type I SMA. A limited study of 23 SMA families also failed to show any influence of APOE genotype on SMA disease severity. Factors other than APOE genotype must therefore be responsible for determining SMA disease severity.

Notechis scutatus venom increases the yield of proliferating muscle cells from biopsies of normal and dystrophic canine muscle--a possible source for myoblast transfer studies.

The injection of 1 micrograms Notechis scutatus (Australian tiger snake) venom (notexin) induces localized necrosis in the muscles of normal and dystrophic dogs. Biopsies taken from the muscles on the second day of postnecrotic regeneration provide about 8-16 x 10(6) cells capable of proliferation per g tissue, about 100 fold more than the untreated adult dog muscles. Muscle specific markers, such as the capacity of the cells to fuse, surface labelling with N-CAM antibodies (Leu-19 and 5.1.H11), and immunostaining with desmin, indicated that over 90% of the cultivated cells are indeed myogenic. The method is a safe and cost effective way to generate large amounts of proliferating muscle cells from biopsies of adult animals, which could provide a useful step in the therapeutic efforts in inherited muscle diseases by the implantation of normal myoblasts or genetically corrected myoblasts.

Expression of dystrophin-associated glycoproteins and utrophin in carriers of Duchenne muscular dystrophy.

The expression of dystrophin, the dystrophin-associated proteins and utrophin has been studied immunocytochemically in three young, manifesting carriers of Duchenne muscular dystrophy, aged 3, 5 and 12 yrs, one adult manifesting carrier, aged 60 yrs, and one presumptive carrier with a raised serum creatine kinase, aged 24 yrs, the mother of the 5-yr-old manifesting carrier. The manifesting carriers had variable degrees of weakness; the presumptive carrier had no weakness. Morphological abnormalities were also variable and were most marked in the young manifesting carriers. The three young manifesting carriers and the presumptive carrier had a mosaic pattern of dystrophin-positive and dystrophin-negative fibres. All the dystrophin-associated proteins were reduced in the dystrophin-deficient fibres, giving a similar mosaic pattern to dystrophin. Expression of dystrophin and the dystrophin-associated proteins was normal in the adult manifesting carrier. Utrophin was detected on the sarcolemma of fibres both with and without dystrophin and the dystrophin-associated proteins. Thus, dystrophin and utrophin are co-expressed in several fibres in carriers. The results emphasize the close association between dystrophin and the glycoprotein complex and their role in the pathogenesis of muscle damage. In addition, the presence of utrophin in fibres with greatly reduced glycoproteins suggests that very little of the glycoprotein complex may be required to anchor the amount of utrophin expressed at the sarcolemma in these particular cases.

Molecular studies of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is inherited as an autosomal recessive disorder which presents as a severe, intermediate or mild condition. The disease selectively affects the alpha motor neuron but nothing is as yet known about the underlying biochemical defect. Recent genetic studies have mapped all three types of SMA to the same region of human chromosome 5 (5q11.2-q13.3) raising the possibility that the mutations may be allelic. Polymorphic DNA markers have been characterised which are suitable for prenatal diagnosis. This is the first step in the isolation of the mutant gene (or genes) involved in this disorder.

Early onset autosomal dominant myopathy with rigidity of the spine: a possible role for laminin beta 1?

We describe 17 individuals from seven families with a slowly progressive, early onset, autosomal dominant myopathy with proximal muscle weakness, calf hypertrophy, contractures, spinal rigidity and, in five of the adult cases, a cardiac conduction defect. A deficiency of the laminin beta 1 chain of the skeletal muscle fibres was found in the older individuals of these families, but not the younger members. Other laminin chains, dystrophin and the dystrophin-associated glycoproteins were normal. The age-related deficiency of the laminin beta 1 is restricted to the skeletal muscle fibres and not the vascular tissue, suggesting that this may be a secondary phenomenon. These findings suggest that a laminin or a laminin-binding protein is implicated in some forms of dominant limb girdle myopathies.

Experimental regeneration in canine muscular dystrophy--1. Immunocytochemical evaluation of dystrophin and beta-spectrin expression.

The expression of dystrophin and beta-spectrin was examined from 1 to 56 days in regenerating muscle fibres in normal and dystrophic dogs, following necrosis induced by the venom of Notechis scutatis. Normal and dystrophic dog muscle regenerated at an equal rate and new myotubes were present in both at the periphery of necrotic fibres by 3 days. In normal dogs dystrophin was detected in the sarcoplasm of the regenerating fibres by 3 days and was localized to the plasma membrane by 4 days. The localization of dystrophin is independent of beta-spectrin and was detected before beta-spectrin, which was not observed until 5-6 days. Normal peripheral labelling of both was restored by 14 days in normal dogs. Normal beta-spectrin labelling of regenerating dystrophic fibres was also restored by 14 days and is not dependent on the presence of dystrophin in dystrophic dogs. A proportion of regenerating fibres in normal and dystrophic dogs showed weak immunolabelling of beta-spectrin prior to 14 days. This is a feature of immature muscle fibres. Antibodies to different domains of dystrophin bound to the periphery and sarcoplasm of regenerating fibres in dystrophic dogs, particularly during the first 7 days of regeneration, but the fluorescence was less intense than in normal dogs. Weak labelling with antibodies corresponding to the C-terminus of the rod domain of dystrophin persisted on dystrophic regenerating fibres up to 21 days. This may relate to developmental isoforms of dystrophin.