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Congenital muscular dystrophy comprises a heterogeneous group of disorders, that have in common an early onset and a dystrophic picture on the muscle biopsy. The "pure" form of congenital muscular dystrophy is not associated with severe mental retardation or structural changes in the brain, though white matter changes on brain imaging have been detected in a significant proportion of cases. In this study we evaluated the incidence of sensory abnormalities (somatosensory and visual evoked responses) in a group of 17 patients with "pure" congenital muscular dystrophy and correlated the results of the evoked responses with the presence or absence of white matter changes on brain magnetic resonance imaging. Our results show close correlation between the presence of MRI white matter changes and abnormalities in the sensory evoked potentials. Conversely, all patients with normal brain MRI had normal somatosensory evoked potentials (SEP). Visual evoked potentials were less sensitive than somatosensory evoked potentials in detecting abnormalities in children with white matter changes on MRI. With the recent discovery of deficiency in merosin expression in the skeletal muscle of a subgroup of patients with CMD, we also correlated the presence or absence of white matter changes and the SEP responses with the merosin status. The results indicate that all merosin-negative patients had abnormal SEP as well as abnormal MRI, whilst no patient with normal merosin expression had an abnormal scan or abnormal SEP.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent studies have identified a group of patients with cytochrome oxidase (COX) deficiency presenting in infancy associated with a deficiency of mtDNA in muscle or other affected tissue (Moraes et al 1991). We used a navel approach to compare the level of mitochondrial (mtDNA) compared to nuclear DNA in skeletal muscle from a group of patients and controls, based on dot blots that were hybridized with a mtDNA probe labelled with S-35[dCTP] and a reference nuclear DNA probe labelled with [P-32]dCTP. The ratio of mtDNA to nuclear DNA varied in samples from different muscles of the same individual. Secondly, fetal muscle had very low levels of mtDNA compared to nuclear DNA, and data from older controls (cross-sectional rather than sequential) suggest that this increases rapidly over the first 3 months after birth and thereafter more slowly. Four patients with COX deficiency had levels of mtDNA that were below the age-specific range defined by 'normal' quadriceps muscle. The clinical features of two of these patients were similar to earlier case reports of mtDNA depletion. Tn three patients the clinical course was relatively benign compared to cases that have previously been described. Levels of mtDNA in skeletal muscle from some patients with other farms of muscle disease were also found to be low, suggesting that mtDNA depletion, possibly related to depletion of mitochondria, may be a relatively non-specific response of muscle to various pathological processes. However, there does appear to be a distinctive group of young patients with reduced cytochrome oxidase activity in muscle, in whom marked mtDNA depletion reflects the primary defect.

Two mutations, skeletal α-actin K326N and β-tropomyosin ΔK7 were reported in patients with ‘stiff’ muscles and spontaneous contractures, respectively, suggesting a hypercontractile phenotype. K326N actin was isolated from a patient biopsy and ΔK7 β-tropomyosin was expressed in baculovirus/sf9 cells. Ca2+ regulation of reconstituted thin filaments was studied by quantitative in vitro motility assay. Both mutations increased Ca2+-sensitivity (EC50 mut/WT = 0.37 ± 0.05 and 0.45 ± 0.25 respectively). In recent models of actin-α-tropomyosin in the OFF state, both actin K326 and tropomyosin K7 are located in the actin-tropomyosin interface. We hypothesise that the charge change due to the mutation destabilises the OFF state and moves the thin filament equilibrium towards the ON state, thus accounting for the higher Ca2+-sensitivity. We also examined a pair of mutations that cause loss of function. Skeletal α-actin D292V (from a patient biopsy) and β-tropomyosin E117K (expressed in baculovirus/sf9 cells) cause congenital fibre type disproportion (hypotonia and weak contractility with small type 1 muscle fibres but without nemaline bodies). In reconstituted thin filaments β-tropomyosin E117K caused a decrease in Ca2+-sensitivity (EC50 mut/WT = 2.44 ± 0.55). Addition of tropomyosin to actin D292V filaments caused a complete switch-OFF of motility that could not be reversed by troponin at high Ca2+ or even NEM-S-1. Thus congenitally weak muscles correlate with loss of function at the molecular level. These mutations are not at the interface of the OFF state but have an opposite charge change to the gain-of-function mutations and are in a location that could destabilise the ON state, which may account for the loss of function.

The secondary dystroglycanopathies are a recessively inherited diverse group of muscular dystrophies ranging in severity from congenital onset with severe cobblestone lissencephaly (Walker-Warburg Syndrome) to milder forms of limb girdle muscular dystrophy (LGMD). Their hallmark is the reduction of alpha-dystroglycan (ADG) glycosylation and an important part of this glycosylation is a unique O-mannosylation which regulates the interaction between ADG and extracellular matrix proteins including laminin alpha 2, perlecan and agrin. The genetic aetiologies of approximately 50% of the dystroglycanopathies have been attributed to nine genes that are involved in ADG glycosylation. Despite collaborative efforts of several international groups using traditional positional cloning, the causative genes for the unsolved dystroglycanopathy cases had escaped discovery. Recently bi-allelic loss of function mutations in the isoprenoid synthase domain containing (ISPD) have been described in Walker-Warburg syndrome. In a collaborative study with the UK10K Rare Disease Project from the Sanger Institute, using whole exome sequencing techniques, we have now discovered that mutations in ISPD are found in a significant proportion of ADG patients. The mutations identified are milder mutations, and the phenotype of affected patients ranges from muscle-eye-brain like disorders, to congenital muscular dystrophy without brain involvement and to limb girdle muscular dystrophy with or without evidence of brain involvement. While in eubacteria and in choloroblasts of higher plants ISPD orthologues are involved in the synthesis of isoprenoid precursors, this pathway is absent in eukaryotes and vertebrates and; therefore, the function of ISPD remains unknown. The mechanism of ISPD mutations leading to reduction of ADG glycosylation needs to be uncovered; however, our ongoing screening shows that ISPD mutations are a common cause of dystroglycanopathies.

Exon skipping using antisense oligonucleotides (AONs) is a promising therapy for Duchenne muscular dystrophy (DMD) which aims to genocopy the milder Becker muscular dystrophy (BMD) by restoring the reading frame of an out-of-frame deletion. The less severe phenotype of BMD patients suggests that internally truncated dystrophin retains some functionality. We previously demonstrated that dystrophin and dystrophin-associated protein expression is correlated to clinical severity in BMD patients with in-frame deletions relevant to current exon skipping strategies (exons 51 and 53), and that BMD patients with deletions ending in exon 51 have significantly higher dystrophin levels than those ending in exon 53. We have now studied deletions relevant for skipping exons 44 and 45 which are now being considered for clinical trials. We studied a total of 25 patients; 11 patients had out-of-frame mutations correctable by skipping exons 44 or 45, and 14 patients had in-frame deletions corresponding to the skipping of exon 45. We quantified dystrophin and dystrophin-associated protein expression using immunohistochemistry and total dystrophin mRNA levels using qRT-PCR. DMD patients skippable for exons 44 and 45 had high levels of revertant and trace dystrophin expression (mean: 14% of control) with 6/11 out-of-frame patients presenting with an intermediate phenotype. Corresponding in frame deletions presented with phenotypes ranging from mild to severe BMD, and showed remarkably lower dystrophin levels (∼42% of control) than patients with deletions modelling the skipping of exons 51 or 53 (80% and 56% of control, respectively). Our study provides valuable insights into the stability and/or function of internally truncated dystrophin suggesting that higher levels of the dystrophin produced by skipping exons 44 and 45 are required to achieve the same level of functionality observed with exon 51 and 53 skipping studies.

P03 What do MHCn and MHCd antibodies recognise? L. Feng, R. Phadke, F. Muntoni, C. Sewry. Dubowitz Neuromuscular Centre, Great Ormond Street Hospital and Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK; UCL Institute of Neurology, National Hospital for Neurology and Neurosurgery, London WC1N 3BG, UK; Centre for Inherited Neuromuscular Disorders, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust, Oswestry, SY10 7AG, UK

Background The currently accepted diagnostic criteria for inclusion body myositis (IBM) comprise pathological and clinical elements. However, the pathological features are not specific to IBM and in many clinically typical cases may be absent. A variety of proteins more commonly associated with neurodegenerative diseases have been found to accumulate in muscle fibres in IBM. The benefit of staining for these proteins in the diagnosis of IBM is unknown. We propose that these protein accumulations may assist in the diagnosis of IBM and help in differentiating it from other myopathies. Aims To identify the most sensitive histochemical technique available to UK diagnostic pathology laboratories for detecting such protein inclusions and to determine their sensitivity and specificity for the diagnosis of IBM. Methods We identified six cases of pathologically definite IBM according to the current criteria and six normal control cases. All cases had been assessed clinically and had undergone a biopsy at the National Hospital for Neurology and Neurosurgery. A number of neurodegenerative proteins and inflammatory markers were identified that may be of diagnostic significance. Antibodies to these proteins were optimised using control tissue known to contain the protein of interest. Initial results Protein inclusions found in IBM were labelled with an antibody to p62, an adaptor protein which binds ubiquitin and regulates signalling cascades through ubiquitination. These inclusions were not found in the normal controls. Further work to quantify the abnormalities in IBM and disease controls will be undertaken before any of these markers can be recommended for diagnostic use.

Deletions in the dystrophin gene give rise to both Duchenne and Becker muscular dystrophies. Good correlation is generally found between the severity of the phenotype and the effect of the deletion on the reading frame: deletions that disrupt the reading frame result in a severe phenotype, while in frame deletions are associated with a milder disease course. Rare exceptions to this rule, mainly owing to frameshift mutations in the 5' region of the gene (in particular deletions involving exons 3 to 7) which are associated with a milder than expected phenotype, have been reported previously. In order to characterise better the relationship between genotype and phenotype as a result of mutations arising in the 5' region of the gene, we have studied a large cohort of patients with small in frame and out of frame deletions in the first 13 exons of the dystrophin gene. Fifty-five patients with a deletion in this area were identified; approximately one third of them had a phenotype different from that theoretically expected. Patients were divided into two groups: (1) patients with a severe clinical phenotype despite the presence of a small, in frame deletion and (2) patients with a mild phenotype and an out of frame deletion. Noticeable examples observed in the first group were Duchenne boys with a deletion of exon 5, of exon 3, and of exons 3-13. In the second group we observed several patients with an intermediate or Becker phenotype and out of frame deletions involving not only the usual exons 3-7 but also 5-7 and 3-6. These data indicate that a high proportion of patients with a deletion in the 5' end of the gene have a phenotype that is not predictable on the basis of the effect of the deletion on the reading frame. The N-terminus of dystrophin has at least one actin binding domain that might be affected by the small, in frame deletions in this area. The effect of the in frame deletions of exon 3, 5, and 3-13 on this domain might account for the severe phenotype observed in these patients. Other mechanisms, such as unexpected effect of the deletion on splicing behaviour, might, however, also be implicated in determining the phenotype outcome.

Recent studies show that patients presenting with cytochrome oxidase (COX) deficiency in infancy may have reduced mitochondrial DNA (mtDNA) in muscle. The human mitochondrial transcription factor A (h-mtTFA) may be an important regulator of both transcription and replication of mtDNA. h-mtTFA levels were investigated in cell lines which were either free of mtDNA (rho 0) or temporarily depleted by treatment with dideoxycytidine (ddC), and in tissue from three patients with mtDNA depletion and cytochrome oxidase deficiency. h-mtTFA was compared with other mitochondrial proteins such as pyruvate dehydrogenase and porin by Western blotting. The ratio of mtDNA and h-mtTFA mRNA to reference nuclear probes was measured by dual labelling of dot blots. The ratio of mtDNA to nuclear DNA in skeletal muscle was low in muscle in the three patients and in other tissues in one. h-mtTFA was low in cells depleted either permanently or transiently of mtDNA, and this reduction in h-mtTFA roughly paralleled mtDNA levels. Similarly, treatment of rho 0 cell lines with ddC induced a reduction in mtDNA as well as h-mtTFA protein. The relationship between h-mtTFA and mtDNA levels suggests that they may be causally linked. MtDNA depletion was accompanied by an increase in the level of h-mtTFA RNA in the cell lines but low levels in the patient. This suggests that either h-mtTFA regulates mtDNA levels, or that h-mtTFA expression may be regulated by a feedback mechanism initiated by MtDNA Depletion.

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.

The regulation of utrophin, the autosomal homologue of dystrophin, has been studied in the canine X-linked model of Duchenne muscular dystrophy. Dystrophic muscle has been shown to exhibit abnormal sarcolemmal expression of utrophin, in addition to the normal expression at the neuromuscular junction, in peripheral nerves, vascular tissues and regenerating fibres. To establish whether this abnormal presence of utrophin in dystrophic muscle is a consequence of continued expression following regeneration, or is attributable to a disease related up-regulation, the expression of utrophin was compared immunocytochemically with that of dystrophin, beta-spectrin and neonatal myosin in regenerating normal and dystrophic canine muscle, following necrosis induced by the injection of venom from the snake Notechis iscutatis. In normal regenerating muscle, sarcolemmal utrophin and dystrophin were detected concomitantly from 2-3 d post-injection, prior to the expression of beta-spectrin. Down-regulation of utrophin was apparent in some fibres from 7 d, and it was no longer present on the extra-junctional sarcolemma by 14 d. Neonatal myosin was still present in all fibres at this stage, but dystrophin and beta-spectrin had been fully restored. In dystrophic regenerating muscle, down-regulation of utrophin occurred from 7 d, although it persisted on some fibres until 28 d, longer than in normal muscle. At 42 d, however, utrophin in dystrophic muscle was only detected in a population of small fibres thought to represent a second cycle of regeneration, with no immunolabelling of mature fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Two male cousins with severe childhood, autosomal recessive. Duchenne-like, muscular dystrophy (SCARMD) have been identified with a deficiency of the 50 kDa dystrophin-associated glycoprotein but normal expression of dystrophin. Both boys were from consanguineous marriages and were Asian, having originated from Pakistan. This is in contrast to all previously reported cases from North Africa. Clinical severity varied and the patients were still able to walk at 13 and 12 yr, respectively. Neither had calf hypertrophy, a feature reported to be almost consistent in the North African patients. Abnormal expression of utrophin, the dystrophin-related protein, was observed on the surface of several non-regenerating muscle fibres, with less intense immunolabelling in the clinically more affected child. This family shows that SCARMD is not confined to North Africa and illustrates a hitherto unreported expression of utrophin in this condition.

The sequential expression of neonatal, fast and slow myosin heavy chain isoforms was examined during the regeneration of normal and dystrophic dog muscle from 1 to 56 days, following necrosis induced by the venom of Notechis scutatis , to assess the regenerative potential of dystrophic muscle. Regeneration was equally rapid in normal and dystrophic dogs but morphological and immunocytochemical abnormalities were more apparent in the dystrophic fibres. New myotubes were formed by 3 days, and by 4 days all myosin isoforms were expressed. Neonatal myosin persisted in normal dogs after morphological restoration of the muscle and after dystrophin and β-spectrin expression had returned to normal. Neonatal myosin was considerably reduced by 21 days in normal dogs, but persisted beyond 28 days in dystrophic dogs, suggesting a delay in maturation. At 42 and 56 days, dystrophic dogs showed a population of small fibres expressing neonatal myosin, which may represent a second cycle of degeneration and regeneration. The reciprocal pattern of fast and slow myosin was not fully restored in normal or dystrophic regenerating muscle, and co-expression persisted. Thus, dystrophic muscle retains its potential to regenerate, but maturation is slower than normal. The persistent co-expression of isoforms has implications for the long-term function of fibres formed after myoblast therapy, but the results imply that a stable state can be achieved if dystrophin expression is restored by gene or myoblast transfer therapy.

The UK MDEX Consortium (http://www.mdex.org.uk/) is involved in close collaboration with AVI BioPharma, in clinical trials using antisense oligonucleotides (AOs) to induce exon skipping in boys with DMD. In 2008 we completed a dose-escalation IM study of a morpholino (PMO) AO, AVI-4658, which induces skipping exon 51 in dystrophin mRNA. In February 2009 we initiated a dose escalation study in ambulant DMD boys aged 5–15 years with deletions benefitting from skipping exon 51. This study consists of 12 weekly administrations of AVI-4658 followed by a muscle biopsy to assess dystrophin expression at baseline and 14 weeks. Clinical parameters are followed for 26 weeks, consisting of safety (adverse events, physical examinations, laboratory tests), muscle, pulmonary and cardiac function, and pharmacokinetics at 1, 6 and 12 doses. A Data Safety Monitoring Board guides dose escalation decisions. Cohorts 1–5 completed 12 weeks of dosing (January 2010), while cohort 6 will complete dosing in March 2010 and clinical observations in June. No drug related SAEs or severe drug related AEs have been reported so far. To date, single doses of 900mg and cumulative exposure exceeding 10,000mg have been well tolerated. Exon skipping and dystrophin protein expression in the cohorts analysed so far indicates a dose response in exon skipping and protein expression in the cohorts up to 4.0mg/kg, with data in the 10.0 and 20.0mg/kg cohorts available soon. These results suggest that AVI-4658 has the potential to lead to the development of a drug that could play a role in the treatment of DMD.

Dystrophin, the product of the Duchenne muscular dystrophy (DMD) gene, was studied in muscle from 16 human fetuses at risk for the disease. Eleven high risk (greater than 95% probability) and 5 low-risk (less than 25% probability) fetuses were studied with antibodies raised to different regions of the protein. All low-risk fetuses showed a similar pattern to that of normal fetuses of a comparable age: using Western blot analysis, a protein was detected of similar size and abundance to that of normal fetuses (i.e. smaller molecular weight than that of adult muscle); immunocytochemistry showed uniform sarcolemmal staining in fetuses older than 18 weeks gestation and differential staining of myotubes at different stages of development (distinguished by size) in younger fetuses (less than 15 weeks gestation). In contrast, Western blot analysis of high-risk fetuses detected low levels of dystrophin in 4 cases; 7 fetuses had no detectable protein. Immunocytochemistry with some dystrophin antibodies showed weak staining of the sarcolemma and around central nuclei in younger fetuses; in older fetuses there was little sarcolemmal staining with any antibody other than occasional positive fibres. These results indicate that careful study of dystrophin in fetuses at risk for DMD can be used to establish the clinical phenotype and provide additional information for future family counselling.

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.

The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and C-terminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to beta-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.

Summary A case of influenza A H 3 N 2 resulting in unusually severe rhabdomyolysis and myoglobinuria is described. Although a rare complication of viral infection, prompt treatment with intravenous fluids can prevent the serious complications which may follow.