Myopathology in congenital myopathies.

Congenital myopathies are clinically and genetically a heterogeneous group of early onset neuromuscular disorders, characterized by hypotonia and muscle weakness. Clinical severity and age of onset are variable. Many patients are severely affected at birth while others have a milder, moderately progressive or nonprogressive phenotype. Respiratory weakness is a major clinical aspect that requires regular monitoring. Causative mutations in several genes have been identified that are inherited in a dominant, recessive or X-linked manner, or arise de novo. Muscle biopsies show characteristic pathological features such as nemaline rods/bodies, cores, central nuclei or caps. Small type 1 fibres expressing slow myosin are a common feature and may sometimes be the only abnormality. Small cores (minicores) devoid of mitochondria and areas showing variable myofibrillar disruption occur in several neuromuscular disorders including several forms of congenital myopathy. Muscle biopsies can also show more than one structural defect. There is considerable clinical, pathological and genetic overlap with mutations in one gene resulting in more than one pathological feature, and the same pathological feature being associated with defects in more than one gene. Increasing application of whole exome sequencing is broadening the clinical and pathological spectra in congenital myopathies, but pathology still has a role in clarifying the pathogenicity of gene variants as well as directing molecular analysis.

Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.

Muscle contraction results from the force generated between the thin filament protein actin and the thick filament protein myosin, which causes the thick and thin muscle filaments to slide past each other. There are skeletal muscle, cardiac muscle, smooth muscle and non-muscle isoforms of both actin and myosin. Inherited diseases in humans have been associated with defects in cardiac actin (dilated cardiomyopathy and hypertrophic cardiomyopathy), cardiac myosin (hypertrophic cardiomyopathy) and non-muscle myosin (deafness). Here we report that mutations in the human skeletal muscle alpha-actin gene (ACTA1) are associated with two different muscle diseases, 'congenital myopathy with excess of thin myofilaments' (actin myopathy) and nemaline myopathy. Both diseases are characterized by structural abnormalities of the muscle fibres and variable degrees of muscle weakness. We have identified 15 different missense mutations resulting in 14 different amino acid changes. The missense mutations in ACTA1 are distributed throughout all six coding exons, and some involve known functional domains of actin. Approximately half of the patients died within their first year, but two female patients have survived into their thirties and have children. We identified dominant mutations in all but 1 of 14 families, with the missense mutations being single and heterozygous. The only family showing dominant inheritance comprised a 33-year-old affected mother and her two affected and two unaffected children. In another family, the clinically unaffected father is a somatic mosaic for the mutation seen in both of his affected children. We identified recessive mutations in one family in which the two affected siblings had heterozygous mutations in two different exons, one paternally and the other maternally inherited. We also identified de novo mutations in seven sporadic probands for which it was possible to analyse parental DNA.

Reversible infantile respiratory chain deficiency is a unique, genetically heterogenous mitochondrial disease.

OBJECTIVES: Homoplasmic maternally inherited, m.14674T>C or m. 14674T>G mt-tRNA(Glu) mutations have recently been identified in reversible infantile cytochrome c oxidase deficiency (or 'benign COX deficiency'). This study sought other genetic defects that may give rise to similar presentations. PATIENTS: Eight patients from seven families with clinicopathological features of infantile reversible cytochrome c oxidase deficiency were investigated. METHODS: The study reviewed the diagnostic features and performed molecular genetic analyses of mitochondrial DNA and nuclear encoded candidate genes. RESULTS: Patients presented with subacute onset of profound hypotonia, feeding difficulties and lactic acidosis within the first months of life. Although recovery was remarkable, a mild myopathy persisted into adulthood. Histopathological findings in muscle included increased lipid and/or glycogen content, ragged-red and COX negative fibres. Biochemical studies suggested more generalised abnormalities than pure COX deficiency. Clinical improvement was reflected by normalisation of lactic acidosis and histopathological abnormalities. The m.14674T>C mt-tRNA(Glu) mutation was identified in four families, but none had the m. 14674T>G mutation. Furthermore, in two families pathogenic mutations were also found in the nuclear TRMU gene which has not previously been associated with this phenotype. In one family, the genetic aetiology still remains unknown. CONCLUSIONS: Benign COX deficiency is better described as 'reversible infantile respiratory chain deficiency'. It is genetically heterogeneous, and patients not carrying the m.14674T>C or T>G mt-tRNA(Glu) mutations may have mutations in the TRMU gene. Diagnosing this disorder at the molecular level is a significant advance for paediatric neurologists and intensive care paediatricians, enabling them to select children with an excellent prognosis for continuing respiratory support from those with severe mitochondrial presentation in infancy.

RYR1 mutations are a common cause of congenital myopathies with central nuclei.

OBJECTIVE: Centronuclear myopathy (CNM) is a rare congenital myopathy characterized by prominence of central nuclei on muscle biopsy. CNM has been associated with mutations in MTM1, DNM2, and BIN1 but many cases remain genetically unresolved. RYR1 encodes the principal sarcoplasmic reticulum calcium release channel and has been implicated in various congenital myopathies. We investigated whether RYR1 mutations cause CNM. METHODS: We sequenced the entire RYR1 coding sequence in 24 patients with a diagnosis of CNM from South Africa (n = 14) and Europe (n = 10) and identified mutations in 17 patients. The most common genotypes featured compound heterozygosity for RYR1 missense mutations and mutations resulting in reduced protein expression, including intronic splice site and frameshift mutations. RESULTS: The high incidence in South African patients (n = 12/14) in conjunction with recurrent RYR1 mutations associated with common haplotypes suggested the presence of founder effects. In addition to central nuclei, prominent histopathological findings included (often multiple) internalized nuclei and type 1 fiber predominance and hypotrophy with relative type 2 hypertrophy. Although cores were not typically seen on oxidative stains, electron microscopy revealed subtle abnormalities in most cases. External ophthalmoplegia, proximal weakness, and bulbar involvement were prominent clinical findings. INTERPRETATION: Our findings expand the range of RYR1-related phenotypes and suggest RYR1 mutations as a common cause of congenital myopathies with central nuclei. Corresponding to recent observations in X-linked CNM, these findings indicate disturbed assembly and/or malfunction of the excitation-contraction machinery as a key mechanism in CNM and related myopathies.

Importance of immunohistochemical evaluation of developmentally regulated myosin heavy chains in human muscle biopsies.

Our retrospective immunohistochemical study of normal quadriceps muscle biopsies shows that embryonic myosin heavy chains are down-regulated by, or soon after, birth. Fetal myosin heavy chains are down-regulated by 4-6 months. Thus the presence of an appreciable number of fibres with embryonic myosin heavy chains at birth or of fetal myosin heavy chains after 6 months of age suggests a delay in maturation or an underlying abnormality. Regenerating fibres in dystrophic muscle often co-express both embryonic and fetal myosin heavy chains but more fibres with fetal than embryonic myosin heavy chains can occur. Embryonic myosin heavy chains are a useful marker of regeneration but effects of denervation, stress, disuse, and fibre maintenance also have to be taken into account. In neurogenic disorders fibres with embryonic myosin heavy chains are rare but fetal myosin heavy chain expression is common, particularly in 5q spinal muscle atrophy. Nuclear clumps in denervated muscle show fetal and sometimes embryonic myosin heavy chains. Developmentally regulated myosins are useful for highlighting the perifascicular atrophy in juvenile dermatomyositis. Our studies highlight the importance of baseline data for embryonic and fetal myosin heavy chains in human muscle biopsies and the importance of assessing them in a spectrum of neuromuscular disorders.

Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression.

AIMS: The quantification of protein levels in muscle biopsies is of particular relevance in the diagnostic process of neuromuscular diseases, but is difficult to assess in cases of partial protein deficiency, particularly when information on protein localization is required. The combination of immunohistochemistry and Western blotting is often used in these cases, but is not always possible if the sample is scarce. We therefore sought to develop a method to quantify relative levels of sarcolemma-associated proteins using digitally captured images of immunolabelled sections of skeletal muscle. METHODS: To validate our relative quantification method, we labelled dystrophin and other sarcolemmal proteins in transverse sections of muscle biopsies taken from Duchenne muscular dystrophy and Becker muscular dystrophy patients, a manifesting carrier of Duchenne muscular dystrophy and normal controls. RESULTS: Using this method to quantify relative sarcolemmal protein abundance, we were able to accurately distinguish between the different patients on the basis of the relative amount of dystrophin present. CONCLUSIONS: This comparative method adds value to techniques that are already part of the diagnostic process and can be used with minimal variation of the standardized protocols, without using extra amounts of valuable biopsy samples. Comparative quantification of sarcolemmal proteins on immunostained muscle sections will be of use to establish both the abundance and localization of the protein. Moreover, it can be applied to assess the efficacy of experimental therapies where only partial restoration or upregulation of the protein may occur.

McArdle disease: a clinical review.

METHODS: The clinical phenotype of 45 genetically confirmed McArdle patients is described. RESULTS: In the majority of patients (84%), the onset of symptoms was from early childhood but diagnosis was frequently delayed until after 30 years of age. Not all patients could recognise a second wind although it was always seen with exercise assessment. A history of myoglobinuria was not universal and episodes of acute renal failure had occurred in a minority (11%). The condition does not appear to adversely affect pregnancy and childbirth. Clinical examination was normal in most patients, muscle hypertrophy was present in 24% and mild muscle wasting and weakness were seen only in patients over 40 years of age and was limited to shoulder girdle and axial muscles. The serum creatine kinase was elevated in all but one pregnant patient. Screening for the mutations pArg50X (R50X) and pGly205Ser (G205S) showed at least one mutated allele in 96% of Caucasian British patients, with an allele frequency of 77% for pArg50X in this population. A 12 min walking test to evaluate patients is described. CONCLUSION: The results demonstrated a wide spectrum of severity with the range of distance walked (195-1980 m); the mean distance walked was 512 m, suggesting significant functional impairment in most patients.

Results of an open label feasibility study of sodium valproate in people with McArdle disease.

McArdle disease results from a lack of muscle glycogen phosphorylase in skeletal muscle tissue. Regenerating skeletal muscle fibres can express the brain glycogen phosphorylase isoenzyme. Stimulating expression of this enzyme could be a therapeutic strategy. Animal model studies indicate that sodium valproate (VPA) can increase expression of phosphorylase in skeletal muscle affected with McArdle disease. This study was designed to assess whether VPA can modify expression of brain phosphorylase isoenzyme in people with McArdle disease. This phase II, open label, feasibility pilot study to assess efficacy of six months treatment with VPA (20 mg/kg/day) included 16 people with McArdle disease. Primary outcome assessed changes in VO2peak during an incremental cycle test. Secondary outcomes included: phosphorylase enzyme expression in post-treatment muscle biopsy, total distance walked in 12 min, plasma lactate change (forearm exercise test) and quality of life (SF36). Safety parameters. 14 participants completed the trial, VPA treatment was well tolerated; weight gain was the most frequently reported drug-related adverse event. There was no clinically meaningful change in any of the primary or secondary outcome measures including: VO2peak, 12 min walk test and muscle biopsy to look for a change in the number of phosphorylase positive fibres between baseline and 6 months of treatment. Although this was a small open label feasibility study, it suggests that a larger randomised controlled study of VPA, may not be worthwhile.

The histopathological spectrum of malignant hyperthermia and rhabdomyolysis due to RYR1 mutations.

OBJECTIVE: The histopathological features of malignant hyperthermia (MH) and non-anaesthetic (mostly exertional) rhabdomyolysis (RM) due to RYR1 mutations have only been reported in a few cases. METHODS: We performed a retrospective multi-centre cohort study focussing on the histopathological features of patients with MH or RM due to RYR1 mutations (1987-2017). All muscle biopsies were reviewed by a neuromuscular pathologist. Additional morphometric and electron microscopic analysis were performed where possible. RESULTS: Through the six participating centres we identified 50 patients from 46 families, including patients with MH (n = 31) and RM (n = 19). Overall, the biopsy of 90% of patients showed one or more myopathic features including: increased fibre size variability (n = 44), increase in the number of fibres with internal nuclei (n = 30), and type I fibre predominance (n = 13). Abnormalities on oxidative staining, generally considered to be more specifically associated with RYR1-related congenital myopathies, were observed in 52%, and included unevenness (n = 24), central cores (n = 7) and multi-minicores (n = 3). Apart from oxidative staining abnormalities more frequently observed in MH patients, the histopathological spectrum was similar between the two groups. There was no correlation between the presence of cores and the occurrence of clinically detectable weakness or presence of (likely) pathogenic variants. CONCLUSIONS: Patients with RYR1-related MH and RM exhibit a similar histopathological spectrum, ranging from mild myopathic changes to cores and other features typical of RYR1-related congenital myopathies. Suggestive histopathological features may support RYR1 involvement, also in cases where the in vitro contracture test is not informative.

Congenital myasthenic syndromes in childhood: diagnostic and management challenges.

The Congenital Myasthenic Syndromes (CMS), a group of heterogeneous genetic disorders of neuromuscular transmission, are often misdiagnosed as congenital muscular dystrophy (CMD) or myopathies and present particular management problems. We present our experience of 46 children with CMS, referred to us between 1992-2007 with provisional diagnoses of congenital myopathy (22/46), CMS or limb-girdle myasthenia (9/46), central hypotonia or neurometabolic disease (5/46), myasthenia gravis (4/46), limb-girdle or congenital muscular dystrophy (4/46) and SMA (2/46). Diagnosis was often considerably delayed (up to 18y4 m), despite the early symptoms in most cases. Diagnostic clues in the neonates were feeding difficulties (29/46), hypotonia with or without limb weakness (21/46), ptosis (19/46), respiratory insufficiency (12/46), contractures (4/46) and stridor (6/46). Twenty-five children had delayed motor milestones. Fatigability developed in 43 and a variable degree of ptosis was eventually present in 40. Over the period of the study, the mainstay of EMG diagnosis evolved from repetitive nerve stimulation to stimulation single fibre EMG. The patients were studied by several different operators. 66 EMGs were performed in 40 children, 29 showed a neuromuscular junction abnormality, 7 were myopathic, 2 had possible neurogenic changes and 28 were normal or inconclusive. A repetitive CMAP was detected in only one of seven children with a COLQ mutation and neither of the two children with Slow Channel Syndrome mutations. Mutations have been identified so far in 32/46 children: 10 RAPSN, 7 COLQ, 6 CHRNE, 7 DOK7, 1 CHRNA1 and 1 CHAT. 24 of 25 muscle biopsies showed myopathic changes with fibre size variation; 14 had type-1 fibre predominance. Three cases showed small type-1 fibres resembling fibre type disproportion, and four showed core-like lesions. No specific myopathic features were associated with any of the genes. Twenty children responded to Pyridostigmine treatment alone, 11 to Pyridostigmine with either 3, 4 DAP or Ephedrine and five to Ephedrine alone. Twenty one children required acute or chronic respiratory support, with tracheostomy in 4 and nocturnal or emergency non-invasive ventilation in 9. Eight children had gastrostomy. Another 11 were underweight for height indicative of failure to thrive and required dietetic input. A high index of clinical suspicion, repeat EMG by an experienced electromyographer and, if necessary, a therapeutic trial of Pyridostigmine facilitates the diagnosis of CMS with subsequent molecular genetic confirmation. This guides rational therapy and multidisciplinary management, which may be crucial for survival, particularly in pedigrees where previous deaths have occurred in infancy.

Multiple mitochondrial DNA deletions in monozygotic twins with OPMD.

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is caused by expansions of the poly (A) binding protein 2 (PABP2) gene. Previous histological analyses have revealed mitochondrial abnormalities in the muscles of OPMD patients but their significance remains uncertain. OBJECTIVE: We had the rare opportunity to study monozygotic twins with identical expansions of the PABP2 gene but with markedly different severities of OPMD. Both had histological features of mitochondrial myopathy. We determined whether mitochondrial DNA abnormalities underlay these changes. METHODS: Clinical information was obtained by history and examination. Muscle biopsies were obtained from each subject and genetic analysis was performed using long-range PCR and Southern blotting. RESULTS: We demonstrate, for the first time, the presence of mitochondrial DNA (mtDNA) deletions by Southern blotting in individuals with OPMD. This correlates with the presence of mitochondrial myopathy in both twins. Moreover, both twins had different mtDNA deletions, which might explain their phenotypic differences. CONCLUSION: We hypothesise that mitochondrial dysfunction may occur as a consequence of PABP2 gene mutations, and that this dysfunction may affect the phenotypic manifestations of OPMD.

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.

A congenital myopathy with diaphragmatic weakness not linked to the SMARD1 locus.

Severe diaphragmatic weakness in infancy is rare. Common causes include structural myopathies, neuromuscular transmission defects, or anterior horn cell dysfunction (spinal muscular atrophy with respiratory distress, SMARD1). We describe a form of infantile diaphragmatic weakness without mutations in the SMARD1 gene, in which pathological and clinical features differ from known conditions, and investigations suggest a myopathy. We identified seven cases in four families. All presented soon after birth with feeding and breathing difficulties, marked head lag, facial weakness, and preserved antigravity movements in the limbs, with arms weaker than legs. All had paradoxical breathing and paralysis of at least one hemi-diaphragm. All required gastrostomy feeding, and all became ventilator-dependent. Investigations included myopathic EMG, muscle biopsy showing myopathic changes, normal electrophysiology and no mutations in SMN1 or IGHMBP2. These seven infants are affected by a myopathic condition clinically resembling SMARD1. However, its pathogenesis appears to be a myopathy affecting predominantly the diaphragm.

Disease severity in dominant Emery Dreifuss is increased by mutations in both emerin and desmin proteins.

Individuals with the same genetic disorder often show remarkable differences in clinical severity, a finding generally attributed to the genetic background. We identified two patients with genetically proven Emery-Dreifuss muscular dystrophy (EDMD) who followed an unusual course and had uncommon clinicopathological findings. We hypothesized digenic inheritance and looked for additional molecular explanations. Mutations in additional separate genes were identified in both patients. The first patient was a member of a family with molecularly proven X-linked EDMD. However, the clinical features were unusually severe for this condition in the propositus: he presented at 2.5 years with severe proximal weakness and markedly elevated serum creatine kinase. Muscle weakness rapidly progressed, leading to loss of independent ambulation by the age of 12. In addition, the patient developed cardiac conduction system disease requiring pacing at the age of 11 and severe dilated cardiomyopathy in the early teens. Despite pacing, he had several syncopal episodes attributed to ventricular dysrhythmias. As these resemble the cardiac features of patients with the autosomal dominant variant of EDMD, we examined the lamin A/C gene, identifying a de-novo mutation in the propositus. The second patient had a cardioskeletal myopathy, similar to his mother who had died more than 20 years previously. Because of the dominant family history, a laminopathy was suspected and a mutation in exon 11 of the LMNA gene was identified. This mutation, however, was not present in his mother, but instead, surprisingly, was identified in his virtually asymptomatic father. Unusual accumulations of desmin found in the cardiac muscle of the propositus prompted us to examine the desmin gene in this patient, and in so doing, we identified a desmin mutation, in addition to the LMNA mutation in the propositus. These cases suggest that separate mutations in related proteins that are believed to interact, or that represent different parts of a presumed functional pathway, may synergistically contribute to disease severity in autosomal dominant EDMD. Furthermore, digenic inheritance may well contribute to the clinical severity of many other neuromuscular disorders.

Muscular dystrophies due to defective glycosylation of dystroglycan.

Muscular dystrophies are a clinically and genetically heterogeneous group of disorders. Until recently most of the proteins associated with muscular dystrophies were believed to be proteins of the sarcolemma associated with reinforcing the plasma membrane or in facilitating its re-sealing following injury. In the last few years a novel and frequent pathogenic mechanism has been identified that involves the abnormal glycosylation of alpha-dystroglycan (ADG). This peripheral membrane protein undergoes complex and crucial glycosylation steps that enable it to interact with LG domain containing extracellular matrix proteins such as laminins, agrin and perlecan. Mutations in six genes (POMT1, POMT2, POMGnT1, fukutin, FKRP and LARGE) have been identified in patients with reduced glycosylation of ADG. While initially a clear correlation between gene defect and phenotype was observed for each of these 6 genes (for example, Walker Warburg syndrome was associated with mutations in POMT1 and POMT2, Fukuyama congenital muscular dystrophy associated with fukutin mutations, and Muscle Eye Brain disease associated with POMGnT1 mutations), we have recently demonstrated that allelic mutations in each of these 6 genes can result in a much wider spectrum of clinical conditions. Thus, the crucial aspect in determining the phenotypic severity is not which gene is primarily mutated, but how severely the mutation affects the glycosylation of ADG. Systematic mutation analysis of these 6 glycosyltransferases in patients with a dystroglycan glycosylation disorder identifies mutations in approximately 65% suggesting that more genes have yet to be identified.

A comparative analysis of collagen VI production in muscle, skin and fibroblasts from 14 Ullrich congenital muscular dystrophy patients with dominant and recessive COL6A mutations.

Ullrich congenital muscular dystrophy (UCMD) is caused by recessive and dominant mutations in COL6A genes. We have analysed collagen VI expression in 14 UCMD patients. Sequencing of COL6A genes had identified homozygous and heterozygous mutations in 12 cases. Analysis of collagen VI in fibroblast cultures derived from eight of these patients showed reduced extracellular deposition in all cases and intracellular collagen VI staining in seven cases. This was observed even in cases that showed normal collagen VI labelling in skin biopsies. Collagen VI immunolabelling was reduced in all the available muscle biopsies. When comparisons were possible no correlation was seen between the extent of the reduction in the muscle and fibroblast cultures, the mode of inheritance or the severity of the clinical phenotype. Mutations affecting glycine substitutions in the conserved triple helical domain were common and all resulted in reduced collagen VI. This study expands the spectrum of collagen VI defects and shows that analysis of skin fibroblasts may be a useful technique for the detection of collagen VI abnormalities. In contrast, immunohistochemical analysis of skin biopsies may not always reveal an underlying collagen VI defect.

Lamin A/C gene mutation associated with dilated cardiomyopathy with variable skeletal muscle involvement.

BACKGROUND: Dilated cardiomyopathy is a form of heart muscle disease characterized by impaired systolic function and ventricular dilation. Familial transmission of the disease is frequently observed, and genetic heterogeneity is indicated by clinical and morphological variability in the disease phenotype. In the family MDDC1 reported here, the disease phenotype is severe and characterized by an autosomal dominant pattern of transmission. In addition, the majority of affected family members show signs of mild skeletal muscle involvement. METHODS AND RESULTS: On the basis of the clinical observation of both cardiac and skeletal muscle abnormalities in the MDDC1 family, the lamin A/C gene was examined in this kindred. Coding regions were polymerase chain reaction-amplified from genomic DNA and sequenced. A single nucleotide deletion was identified within exon 6, and all affected individuals were found to be heterozygous for this deletion. CONCLUSIONS: Heterozygosity for a single nucleotide deletion in exon 6 of lamin A/C segregates with both the cardiac and skeletal abnormalities observed in the MDDC1 family.

A and B utrophin in human muscle and sarcolemmal A-utrophin associated with tumours.

Utrophin is an autosomal homologue of dystrophin, abnormal expression of which is responsible for X-linked Duchenne and Becker muscular dystrophy. In normal mature muscle utrophin is confined to blood vessels, nerves and myotendinous and neuromuscular junctions. When dystrophin is absent utrophin is abundant on the sarcolemma. This has raised the possibility that up-regulation of utrophin may be of therapeutic benefit. Two full-length transcripts of utrophin, A and B, have been identified, which are regulated by alternatively spliced 5' promoters. In dystrophic mouse muscle, the A isoform is present on the sarcolemma, whereas the B form is confined to blood vessels. We show here using immunohistochemistry and human isoform-specific antibodies that A- and B-utrophin localisation is the same in human muscle. The A isoform is present on the sarcolemma of foetal human muscle fibres, regenerating fibres, fibres deficient in dystrophin and on blood vessels and neuromuscular junctions. B-utrophin is only detected on blood vessels. We also show that muscle adjacent to some soft tissue tumours shows increased sarcolemmal utrophin-A, showing that utrophin and dystrophin can simultaneously localise to the sarcolemma and raising the possibility that factor(s) from the tumour cells or accompanying inflammatory cells may have a role in regulating utrophin.

Investigating sodium valproate as a treatment for McArdle disease in sheep.

McArdle disease is due to an absence of the enzyme muscle glycogen phosphorylase and results in significant physical impairment in humans. We hypothesised that sodium valproate, an HDAC inhibitor, might have the ability to up-regulate the enzyme. We treated McArdle sheep with sodium valproate given enterically at 20-60 mg/kg body wt. Compared with untreated control animals, there was increased expression of phosphorylase in muscle fibres. The response was dose dependent and reached a maximum 2 hours after the application and increased with repeated applications. Improvement in mobility could not be demonstrated. These findings suggest that sodium valproate is a potential therapeutic treatment for McArdle disease.

Zebra body myopathy is caused by a mutation in the skeletal muscle actin gene (ACTA1).

We present follow up data on the original case of 'zebra body myopathy' published by Lake and Wilson in 1975. Pathological features in a second biopsy performed at the age of 29 years included a wide variation in fibre size, multiple split fibres, excess internal nuclei and endomysial connective tissue, rimmed vacuoles, accumulation of myofibrillar material and large 'wiped out' areas lacking stain for oxidative enzymes. The presence of nemaline rods and actin-like filaments in addition to small zebra bodies suggested ACTA1 as a candidate gene. This has been confirmed by the identification of a novel c.1043T.p.Leu348Gln mutation, which probably occurred de novo. This case illustrates that the myopathy associated with zebra bodies is part of the spectrum of myopathies associated with the ACTA1 gene. It also highlights that accumulation of actin filaments is not confined to severe neonatal ACTA1 cases and that progression of weakness can occur in congenital myopathies, as the patient is now wheelchair bound and can only stand with the aid of a walking frame.