News in Muscular Dystrophy

Christopher F. Spurney  Susan Knoblach , Emidio E. Pistilli ,Kanneboyina Nagaraju , Gerard R. Martin , Eric P. Hoffman - USA

Duchenne muscular dystrophy (DMD; dystrophin-deficiency) causes dilated cardiomyopathy in the second decade of life in affected males. We studied the dystrophin-deficient mouse heart (mdx) using high-frequency echocardiography, histomorphometry, and gene expression profiling. Heart dysfunction was prominent at 9–10 months of age and showed significantly increased LV internal diameter (end systole) and decreased posterior wall thickness. This cardiomyopathy was associated with a 30% decrease in shortening fraction. Histologically, there was a 10-fold increase in connective tissue volume (fibrosis). mRNA profiling with RT-PCR validation showed activation of key pro-fibrotic genes, including Nox4 and Lox. The Nox gene family expression differed in mdx heart and skeletal muscle, where Nox2 was specifically induced in skeletal muscle while Nox4 was specifically induced in heart. This is the first report of an altered profibrotic gene expression profile in cardiac tissue of dystrophic mice showing echocardiographic evidence of cardiomyopathy.

Larry W. Markham, Kathi Kinnett, Brenda L. Wong, D. Woodrow Benson, Linda H. Cripe  - USA

Duchenne muscular dystrophy (DMD) is characterized by a predictable decline in cardiac function with age that contributes to early death. Although corticosteroids are a clinically effective pharmacologic therapy for skeletal muscle function, there is limited published work documenting the impact on cardiac function. The primary objective of this work is to determine benefit from steroid treatment on the development of ventricular dysfunction in DMD. We performed a historical cohort study of DMD cases undergoing serial cardiac evaluations from 1998–2006. In addition to the history of steroid use, basic medical characteristics and serial echocardiographic measures were obtained for each identified case meeting inclusion criteria. Data from initial (7.5 ± 0.8 years) and follow-up (12 ± 0.7 years) evaluation was collected from untreated (n = 23) and steroid treated (n = 14) DMD cases. Kaplan–Meier freedom from ventricular dysfunction was 93% for steroid treated cases versus 53% for untreated cases at 1500 days. Treatment with steroids was protective against ventricular dysfunction (Hazard ratio 0.16 95% CI 0.04, 0.70). We demonstrate here that steroid treatment, begun prior to ventricular dysfunction retards the anticipated development of ventricular dysfunction.

16 - (Abstracts of the P2T Congres de Physiologie,de Pharmacologie et de Therapeutique, 2008 - Fundamental & Clinical Pharmacology, 22:1-102, 2008) Effect of L-arginine, a NOS substrate, on mdx mouse diaphragm

K Hnia, J Gayraud, A Lacampagne, G Hugon, M Ramonatxo, D Mornet, S Matecki  - France

Introduction: L-arginine was proposed as a pharmacological tool in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disease due to a mutation in the dystrophin gene. Administration of L-arginine (the substrate of nitric oxide synthase) promotes a better muscle membrane integrity in mdx mice, the animal model of the muscle dystrophin-deficiency. Despite the beneficial effect on L-arginine on muscle weakness and force, mechanisms by each this molecule acts on muscle remain poorly investigated. Methods: Five-week-old Male dystrophin-deficient Animals (mdx control; n = 7 and mdx treated; n = 7) were treated for 2 weeks with intraperitoneal injections. Control group was injected with (Ss) physiological saline solution (mdx-Ss). Treated group was injected with L-arginine, (Sigma), 20 ll/vol/g at a Cc of 200 mg/kg (mdx-L-arg). After beheading, the diaphragm was dissected into 2 parts. One part was immediately frozen in liquid nitrogen-cooled isopentane and stored at -80C and the other part was used freshly to determine enzymatic activities, Ca2+-spark’s properties and resistance to eccentric contractions. Results: Here, we showed that L-arginine improve RyR gating mechanisms with a decreased inflammatory secreted cytokine IL-1alpha, IL-6 and TNFalpha targeted to dystrophic muscle fibres. This leads to decrease level and activity of NF-kB and its targeted proteins such as the muscle specific metalloproteinase MMP-2 and MMP-9 which are responsible of the betadystroglycan cleavage into a ~30 kDa form in mdx muscles. The betadystroglycan is a key transmembrane glycoprotein of the dystrophin glycoprotein complex which anchors utrophin to sarcolemma. Conclusion: Our results demonstrate that L-arginine administration promotes a better membrane stability of beta-dystroglycan/utrophin couple and re-localizes nNOS in subsarcolemmal compartment of the dystrophic fibres which leads to improve dystrophic pattern in mdx diaphragm with increase resistance to contraction-induced mechanical stress. Our data strengthen the usefulness of L-arginine as a powerful pharmacological tool in Duchenne muscular dystrophies and also enlarge its use in other muscle-inflammation mediated myopathies.

Sophie Mavrogeni, Antigoni Papavasiliou, Marouso Douskou, Genovefa Kolovou, Evangelia Papadopoulou, Dennis V. Cokkinos - Greece.

Objective: To evaluate the involvement of cardiac and sternocleidomastoid muscles by magnetic resonance imaging (MRI) measurement of T2 relaxation time and the left ventricular systolic function in patients with Duchenne muscular dystrophy (DMD) on treatment with deflazacort and compare them with DMD patients without treatment. Subjects: Seventeen patients with DMD (aged 17–22 years) on treatment with deflazacort for at least 7 years and 17 boys with DMD of younger age (12–15 years) without steroid treatment. All patients were free of cardiac or respiratory symptoms and had normal ECG and Holter monitor examination. Methods: T2 relaxation time of the myocardium (H), left (SCM-L) and right sternocleidomastoid (SCM-R) muscles and left ventricular systolic function were evaluated by magnetic resonance imaging (MRI) in two groups of DMD patients. Myocardial and sternocleidomastoid muscles T2 relaxation time was calculated using 16 TEs (10–85 msec) and TR at least 2000ms and T2 maps were created. Results: DMD on deflazacort had higher T2 relaxation time values of the heart and of both sternocleidomastoid muscles (T2H median (range): 47 (41–48) vs. 33 (31–37) ms, po0.001, T2 SCM-L median (range): 35 (30–37) vs. 23 (20–26) ms, po0.001, T2 SCM-R median (range): 35 (32–37) vs. 23 (20–27) ms, po0.001) and left ventricular systolic function (LVEDV median (range): 95 (75–120) vs. 90 (80–105) ml, p ¼ 0.03, LVESV median (range): 45 (38–55) vs. 47 (41–51) ml, p ¼ 0.81(NS), LVEF median (range): 53% (51–57) vs. 48% (42–51), po0.001) compared to DMD without treatment. Conclusions: DMD patients on deflazacort are characterized by better preservation of the T2 relaxation time of myocardium and sternocleidomastoid muscles and better LV systolic function. The duration of this beneficial effect needs to be studied prospectively.

12 - (Journal of the American Society of Echocardiography, 2008)  Early Regional Myocardial Dysfunction in Young Patients With Duchenne Muscular Dystrophy

Luc Mertens, Javier Ganame, Piet Claus, Nathalie Goemans, Daisy Thijs, Bénédicte Eyskens, David Van Laere, Bart Bijnens, Jan D'hooge, George R. Sutherland, Gunnar Buyse

Background: In young patients (aged <12 years) with Duchenne muscular dystrophy (DMD), cardiac systolic function is generally described to be within the normal range. Recent studies have suggested the presence of subclinical dysfunction in these young patients using cardiac magnetic resonance imaging, tissue Doppler measurements, and myocardial velocity gradients. The aim of this study was to further assess regional myocardial function in a young group of patients with DMD using myocardial velocity and deformation imaging. Methods: Thirty-two patients with DMD (mean age, 7.9 years; range, 3-12 years) and 29 age-matched normal controls were studied with echocardiography. Standard echocardiographic measurements of left ventricular (LV) systolic and diastolic function were performed. Myocardial velocity and deformation data, including peak systolic and early and late diastolic myocardial velocities, peak systolic strain rate (SR), and peak systolic strain (ε), were calculated in the radial direction in the inferolateral LV wall and in the longitudinal direction in the interventricular septum, the LV anterolateral wall, and the right ventricular (RV) free wall. Results: Higher heart rates and increased LV end-systolic dimensions were seen in patients with DMD compared with controls. Significant decreases in radial and longitudinal peak systolic SR, peak systolic ε, and peak systolic and early diastolic myocardial velocities were found in the LV inferolateral and anterolateral walls in patients with DMD. No significant differences in longitudinal function could be found in the interventricular septum or in the RV free wall. Conclusion: In young patients with DMD who have global normal systolic function, reductions in systolic deformation parameters as well as reduced early diastolic myocardial velocities can be detected in the anterolateral and inferolateral LV walls. The prognostic significance of these findings warrants further longitudinal follow-up.

12 - Health Care Utilization and Expenditures for Children and Young Adults With Muscular Dystrophy in a Privately Insured Population

30 - (Neuromuscular Disorders, 2008) Reduced muscle necrosis and long-term benefits in dystrophic mdx mice after cV1q (blockade of TNF) treatment

Hannah G. Radley, Marilyn J. Davies, Miranda D. Grounds  - Australia

Tumour necrosis factor (TNF) is a potent inflammatory cytokine that appears to exacerbate damage of dystrophic muscle in vivo. The monoclonal murine specific antibody cV1q that specifically neutralises murine TNF demonstrated significant anti-inflammatory effects in dystrophic mdx mice. cV1q administration protected dystrophic skeletal myofibres against necrosis in both young and adult mdx mice and in adult mdx mice subjected to 48 h voluntary wheel exercise. Long-term studies (up to 90 days) in voluntarily exercised mdx mice showed beneficial effects of cV1q treatment with reduced histological evidence of myofibre damage and a striking decrease in serum creatine kinase levels. However, in the absence of exercise long-term cV1q treatment did not reduce necrosis or background pathology in mdx mice. An additional measure of well-being in the cV1q treated mice was that they ran significantly more than control mdx mice.

30 - (Developmental Medicine & Child Neurology, Volume 50, Issue s113, 49-59, 2008) PEDIATRIC PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY

Fiona Adams, Susan Bostock, Angela Potter - Australia

Objective: This review aims to give recommendations for best respiratory management (and associated costs) for pediatric patients with Duchenne Muscular Dystrophy, in accordance with evidence in the literature and usual practice in Australia and overseas.

Design: Narrative review of the literature, and a survey of usual practice.

Method: Database search using Medline, CINAHL, PubMed, Cochrane and PEDro from 2003–2007. A survey was conducted amongst paediatric physiotherapists (and 2 physiotherapists working with adults), seeking information (including protocols) on usual management. Liaison with professionals regarding cost and training for recommended interventions also took place.

Results: The interventions supported by the strongest current evidence include mechanical in-exsufflation (MI-E), intrapulmonary percussive ventilation (IPV), nocturnal noninvasive ventilation (NIV), manually assisted cough (MAC), breath- stacking, glossopharyngeal breathing (GPB) and mechanical insufflation. The use of PEP treatment, chest physiotherapy, autogenic drainage and IPPB are supported by low-level evidence, while suction has little and conflicting evidence. Survey findings and protocols indicate majority of practitioners use postural drainage, percussion, MAC, manual insufflation, MI-E, suction and NIV, yet very few include IPV in management of clients with DMD. Technique application is variable depending on patient and physiotherapist. Implementation of MI-E is costly but time efficient. Conclusion: There remains a lack of strong evidence for certain techniques commonly used for children with DMD, such as postural drainage, percussion and suction. Due to strong evidence showing benefit, MI-E , MAC and nocturnal NIV should be continued in practice, while IPV, breath stacking, GPB and mechanical insufflation should be re-considered for implementation (if not already used).

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder of striated muscle caused by mutations in the DMD gene. A recombinant adeno-associated virus (AAV)-mediated gene transfer is one of attractive approaches to the treatment of DMD. To examine therapeutic effects and the safety issue of the approach in larger animal models, we recently established a Beagle-based dystrophic dog colony in Japan, CXMDJ. First, we injected a recombinant AAV2 encoding the LacZ gene (rAAV2- CMVLacZ) into skeletal muscles of normal Beagles. b-galactosidase (b-gal) was expressed only in a few fibers, together with marked cellular infiltration. Immunosuppressants improved b-gal expression, though the effect was not complete (Gene Ther. 14:1249–1260, 2007). We, then, generated a type 8 recombinant AAV (rAAV8)-CMVLacZ, and injected it into skeletal muscle of normal Beagles. Importanly, we found more b-galpositive fibers in AAV8-injected muscles than in AAV2-injected muscles. We, therefore, injected recombinant AAV8 encoding a canine microdystrophin (cDCS1) gene, a truncated version of the dystrophin gene into skeletal muscle of CXMDJ and confirmed widespread expression of micro-dystrophin at the sarcolemma. We also demonstrate that the administration of recombinant AAV8 encoding DCS1 through limb perfusion is a feasible approach to DMD.

Muscular dystrophies comprise a diverse group of genetic disorders that lead to muscle wasting and, in many instances, premature death. Many mutations that cause muscular dystrophy compromise the support network that connects myofilament proteins within the cell to the basal lamina outside the cell, rendering the sarcolemma more permeable or leaky. Here we show that deletion of the gene encoding cyclophilin D (Ppif) rendered mitochondria largely insensitive to the calcium overload–induced swelling associated with a defective sarcolemma, thus reducing myofiber necrosis in two distinct models of muscular dystrophy. Mice lacking -sarcoglycan (Scgd^-/- mice) showed markedly less dystrophic disease in both skeletal muscle and heart in the absence of Ppif. Moreover, the premature lethality associated with deletion of Lama2, encoding the -2 chain of laminin-2, was rescued, as were other indices of dystrophic disease. Treatment with the cyclophilin inhibitor Debio-025 similarly reduced mitochondrial swelling and necrotic disease manifestations in mdx mice, a model of Duchenne muscular dystrophy, and in Scgd^-/- mice. Thus, mitochondrial-dependent necrosis represents a prominent disease mechanism in muscular dystrophy, suggesting that inhibition of cyclophilin D could provide a new pharmacologic treatment strategy for these diseases.

R A Kley, M A Tarnopolsky, M Vorgerd - Canada

Creatine is a naturally occurring amino acid derivative that is obtained by endogenous synthesis and dietary intake. Creatine phosphate has a high phosphoryl group transfer potential and serves as an ATP buffer during muscle contraction. Creatine supplementation increases muscle strength in healthy persons.

CONCLUSIONS

There is evidence from randomised controlled trials that short and medium term creatine treatment improves muscle strength, increases lean body mass and is well tolerated in patients with muscular dystrophies. The available evidence does not support.the use of creatine in metabolic myopathies. It is noteworthy that high dose creatine supplementation increased muscle pain in glycogenosis type V. There is a need for long term creatine studies in muscular dystrophies and creatine trials in hereditary myopathies that have not been targeted for evaluation so far such as myofibrillar myopathies and fatty acid oxidation defects. Furthermore, the effect of creatine supplementation on activities of daily living has to be evaluated.

15 - Standards of care for Duchenne muscular dystrophy - Brief TREAT-NMD recommendations

Sylvie Houde, Michèle Filiatrault, Anne Fournier, Julie Dubé, Sylvie D’Arcy, Denis Bérubé, Yves Brousseau, Guy Lapierre  and Michel Vanasse - Canada

Data reported here were collected over an 8-year period for 79 Duchenne muscular dystrophy patients, 37 of whom were treated with deflazacort. Mean length of treatment was 66 months. Treated boys stopped walking at 11.5 + 1.9 years, compared with 9.6 + 1.4 years for untreated boys. Cardiac function was better preserved with the use of deflazacort, as shown by a normal shortening fraction in treated (30.8 + 4.5%) vs untreated boys (26.6 + 5.7%, P < 0.05), a higher ejection fraction (52.9 + 6.3% treated vs 46 + 10% untreated), and lower frequency of dilated cardiomyopathy (32% treated vs 58% untreated). Scoliosis was much less severe in treated (14 + 2.5°) than in untreated boys (46 + 24°). No spinal surgery was necessary in treated boys. Limb fractures were similarly frequent in treated (24%) and untreated (26%) boys, but vertebral fractures occurred only in the treated group (7/37) (compared with zero for the untreated group). In both groups, body weight excess tripled between the ages of 8 and 12 years. All untreated patients grew normally (>4 cm/year), as opposed to only 15% of treated boys. Deflazacort improves cardiac function, prolongs walking, and seems to eliminate the need for spinal surgery, although vertebral fractures and stunted growth occur. The overall impact on quality of life appears positive.

Duchenne muscular dystrophy (DMD), a common inherited myopathy that affects approximately 1 in 3,500 males, is characterized by progressive muscle wasting due to a deficiency in muscle dystrophin. DMD progresses with a rather uniform pattern of muscle weakness; i.e., DMD causes affected individuals to lose their ability to walk by the age of 12 y, and patients succumb during their twenties due to either respiratory or cardiac failure. The deficiency in dystrophin is caused by translational reading frame shift or nonsense mutations in the dystrophin gene [1]. However, the existence of a modifying gene has been suggested by the identification of unusually mild DMD phenotypes [2-4]. Although some phenotypic variability may arise due to environmental factors, such as diet or exercise, genetic components are likely to contribute to this variability. Myostatin, also known as growth and differentiation factor 8 (GDF8), is a muscle-specific secreted peptide that limits muscle growth [5,6]. However, genotyping of the myostatin gene failed to disclose any nucleotide changes that behaved as a phenotypic modifier of DMD [7]. Remarkably, blocking endogenous myostatin has been shown to result in anatomic, biochemical, and physiologic improvements in the dystrophic phenotype of mdx mice, a mouse model for DMD, including particularly prominent enlarged fiber diameters and greatly reduced fatty fibrosis [8-10]. These results suggest that blocking endogenous myostatin is a potential strategy to treat DMD [11]. We examined the hypothesis that serum myostatin is increased in DMD, thereby enabling treatment by myostatin blockage. Forty-two DMD patients followed at Kobe University Hospital were enrolled in this study. All but 1 of the mutations in the dystrophin gene were found to introduce premature stop codons in the dystrophin mRNA: 24 cases with mutations that induced a translational reading frame-shift due to exon deletion or duplication; 5 cases with nonsense mutations; 7 cases with exon mutations involving one or a few nucleotides deleted or inserted; 3 cases with intron mutations that induced splicing errors; and one case with an abnormal chromosome (Table 1). The subjects’ ages ranged from 1 to 22 y (average: 8.3 y). Regular clinical checkups, including determination of serum creatine kinase (CK) concentrations, were performed at the outpatient clinic. All protocols were approved by the ethics committee of the Kobe University School of Medicine. Blood samples were taken after written informed consent was obtained from all patients, and serum was separated using a clinical centrifuge. Serum myostatin was measured using the Human Myostatin ELISA (Prodomain Specific) kit purchased from BioVendor Laboratory Medicine, Inc. (Bmo, Czech Republic). The upper limit of determination was 50 ng/ml, and normal adults have serum concentrations of 0.19 to 9.02 ng/ml (BioVendor Laboratory Medicine, Inc.). The Pearson product-moment coefficient was calculated to quantify the relationship. Serum myostatin concentrations in DMD patients ranged from 1.1 to >50 ng/dl (Table 1). Remarkably, 13 samples were >50 ng/ml, and the lowest concentration was 1.1 ng/ml (Table 1). Though age differences were examined in 2 conditions either including or deleting 13 samples with >50 ng/ml, no significant correlationship between age and serum myostatin concentration was found (Fig. 1). We next examined whether concentrations of myostatin were related to the type or location of mutations in the dystrophin gene. Though serum myostatin concentrations were compared based on their mutation types (exon deletion/duplication or others), no clear difference between two groups was revealed (Fig. 1). There found no significant difference in serum myostatin concentration between patients with mutation in the 5’ and 3’ regions of the dystrophin gene (Table 1). Considering that myostatin is an inhibitor of muscle growth, cases with high serum myostatin concentrations were predicted to present rather severe phenotypes. Transgenic overexpression of myostatin in mice was shown to result in cachexia [12]. However, the ages when DMD patients with high myostatin concentrations became wheelchair-bound were not different from those of patients with low myostatin concentrations, and signs of muscle weakness appeared mostly between ages 4 and 5. Furthermore, serum CK concentrations were not significantly lower in DMD cases with high myostatin concentrations than in those with low concentrations (Table 1). This indicates that serum myostatin did not modify the DMD phenotype even though blocking endogenous myostatin has been shown to result in improvements in the dystrophic phenotype of mdx mice [8-10]. In this study, we measured myostatin that reacted with a monoclonal antibody recognizing the prodomain of myostatin. Considering that myostatin is secreted as an inactive propeptide and is cleaved to produce the active form, further studies would be required to measure active or latent myostatin individually. Expression of the myostatin gene has been examined previously in skeletal muscle by measuring mRNA and protein concentrations [13]. Serum myostatin concentration has been determined by the Western blot analysis in a patient with a mutation in the myostatin gene, disclosing an absence of myostatin [14]. Furthermore the Western blot analysis disclosed that serum myostatin concentration was lower than that of rat [14]. But no further study has been conducted on serum myostatin concentrations. Our results disclosed a wide range in serum myostatin concentrations in DMD patients. Though myostatin blockage is attracting attention as a novel target for increasing muscle growth in cases of DMD [11], our results suggest that myostatin blockage therapy would only be effective in DMD cases involving high serum myostatin concentrations. Therefore, myostatin blockage therapy should be applied carefully as a treatment for DMD.

Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-a receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.

M KINALI MD MRCPCHA, V ARECHAVALA-GOMEZA MPHARM MSC PHDA, L FENG PHDA, A GLOVERB, M GUGLIERI MDC, H JUNGBLUTH MD PHD MRCP MRCPCHD, H ROPER FRCPCHE, RM QUINLIVAN FRCP FRCPCHF, D HUNT FRCSG, AY MANZUR FRCPCHA, A HENDERSON PHDC, J GOSALAKKALH, K HOLLINGSWORTH PHDI, J ALLSOP DCR(R)J, E MERCURI PHDA,K, J MORGAN PHDA, D J WELLS PHD MRCVSL, C SEWRY PHD FRCPATHA,F, V STRAUB MD PHDC, K BUSHBY MB CHB MSC MD FRCPC, M RUTHERFORD MD FRCR FRCPCHJ, F MUNTONI FRCPCH FMEDSCA - UK

Background: The progressive course of the muscle weakness in Duchenne muscular dystrophy (DMD) has been documented by several, exhaustive natural history studies; however, the progression of muscle pathology with time in individual muscle groups is poorly defined. There are currently no effective treatments to halt the muscle breakdown in DMD, although there are planned clinical trials using gene-based techniques. Most of these techniques, such as antisense oligonucleotides (AON), will require a sufficiently well preserved muscle to be effective. The choice of the muscles to be studied and the role of non-invasive methods to assess muscle preservation, therefore, require further evaluation. Method: Our principal objective was to study the degree of muscle involvement in the lower-leg muscles of 22 patients with DMD aged >8 years, using non-invasive imaging techniques (muscle MRI). In a subgroup of eight patients we also correlated the muscle MRI findings with the histology of muscle biopsies obtained during surgery from a foot muscle, the extensor digitorum brevis (EDB). Muscle MRI involvement was assigned using a 1 to 4 scale (normal–severe involvement). All patients had a gradient of involvement of their lower-leg muscles: posterior compartment (gastrocnemius>soleus) was most severely affected. Anterior compartment (tibialis anterior/posterior, popliteus, extensor digitorum longus) was least affected. Results: Muscle MRI showed a progressive involvement of the EDB but this did not directly correlate with age or time since loss of independent ambulation. There was a good correlation between the MRI and the EDB histopathology, with MRI grades 3 to 4 associated with a more fibro-adipose tissue replacement. Conclusion: Our results indicate that the EDB was sufficiently preserved even in non-ambulant patients and that MRI grading 2 to 3 is associated with sufficiently preserved structure. This information will allow patient recruitment in future clinical trials using AON based on the severity of the involvement of muscle on MRI without necessarily having to verify the muscle preservation invasively.

B) Long-term use of intermittent low-dosage prednisolone therapy in Duchenne muscular dystrophy: tolerance and effect on functional outcome

C POULTON MRCPCHA, M KINALI MD MRCPCHA, S A ROBB MD FRCP FRCPCHA, M MAIN MA MCSPB, AY MANZUR FRCPCHA, F MUNTONI FRCPCH FMEDSCA - UK

We reviewed functional outcome and steroid tolerance in 51 males with Duchenne muscular dystrophy (DMD) treated from 2000 onwards with intermittent prednisolone (0.75mg/kg/ day 10 days on/20 days off, or 10 days on/10 days off) for 18 months or more and compared this with natural history motor ability scores (MAS) previously published from this centre. Twentyseven out of 51 patients remained ambulant (Group 1) and 24 of 51 lost ambulation (Group 2). Regimes and ages at commencement were similar: Group 1 mean age 7 years (range 3y 10mo–11y 8mo); Group 2: mean age 7y 6mo (range 4–11y). Mean prednisolone duration was 2 years 7 months (Group 1) and 3 years 4 months (Group 2). Group 1 had better MAS than the natural history agematched patients (p<0.001) at the outset with a mean MAS 30. ANOVA tests showed stabilization of MAS for 24 months. Group 2 was similar to the natural history patients at outset with mean MAS <30, dropping to 23 after 24 months, a significant variance (p=0.069). Mean age of loss of independent ambulation was 10 years 5 months. Mean age of the still ambulant population was 10 years. Six males remained ambulant beyond 12 years. Age of loss of ambulation did not correlate with the MAS at onset. Forced vital capacity was >70% at 24 months after commencing therapy (n=23). Both steroid regimes were tolerated well. Six patients discontinued therapy, due to weight gain, epigastric pain (n=4), and parental choice (n=1). Behavioural changes data were available for 36 of 51 patients. Six had moderate changes, two required therapy modification. One child, with hydrocephalus, sustained a vertebral fracture. Eleven of 51 patients developed mild cardiomyopathy and two of 51 developed mild scoliosis. This open study demonstrates that in DMD, an intermittent prednisolone regime has a positive, though limited functional benefit in prolonging ambulation and preserving forced vital capacity, without major side effects. Use of the intermittent regime may be helpful to assess the likelihood of side effects, before transition to a daily regime, or may be continued with some benefit, especially if concerned about side effects.

C) Feeding difficulties in Ullrich congenital muscular dystrophy

N MCSWEENEY, A ALOYSIUS, T DAVIS, F MUNTONI - UK

Background: Ullrich congenital muscular dystrophy (UCMD) is an inherited progressive disorder due to a deficiency in Collagen VI, characterized by early onset hypotonia, contractures, distal joint laxity, spinal rigidity, and respiratory insufficiency. Feeding difficulties are frequently observed and patients might require dietary intervention or insertion of a gastrostomy tube. Objectives: To determine: (1) the incidence, (2) the severity of feeding difficulties, and (3) the outcome of intervention in UCMD. Methods: Medical notes of patients followed up at the Dubowitz Neuromuscular Centre with an established diagnosis of UCMD were reviewed. Results:We reviewed 38 case notes of patients with a clinical diagnosis of UCMD (28 males, 10 females, age range 3y 6mo– 32y), established by immunocytochemistry and/or genetic studies. Twenty-four had feeding difficulties and 14 did not. Feeding difficulties were defined as difficulty chewing, dysphagia with liquids or solids, prolonged mealtimes (>30 min), or having a poor appetite. Mean age at onset of feeding difficulties was age 13 years 6 months (median 14y, range birth–30y). Four patients had gastroesophageal reflux, which was treated medically. Faltering growth (weight <0.4th centile) was documented in 16 patients who had feeding difficulties. Fourteen patients received nutritional supplements, which only resulted in weight gain in three. The two patients who had nasogastric feeding had improved weight gain and three of the five who had gastrostomy had improved weight gain. Clinical course was generally more severe in those with feeding difficulties, with increased incidence of lower respiratory tract infections, non-invasive ventilation (mean age 15y), and scoliosis. Conclusion: Feeding difficulties are common in UCMD and broadly correlate with the overall disease severity. Dietary intervention with oral nutritional supplements seems to have little impact on faltering growth. Intervention using enteral feeding was more successful and suggests that intervention with gastrostomy should be considered early in the course of this progressive disorder.

D) Sibling adjustment in Duchenne muscular dystrophy

J READ BSC RGN, M KINALI PHD, F MUNTONI FMEDSCI FRCPCH, ME GARRALDA MD FRCPSYCH FRCPCH - UK

Objective: To document psychosocial adjustment in siblings of males with Duchenne muscular dystrophy (DMD). Methods: Participants were siblings of patients with DMD attending a specialist centre, aged 11 to 18 years, with no physical problems. We used quantitative measures: parent rated sociodemographic and illness questionnaires and interviews, and sibling self-rated questionnaires on physical and mental health (SF-36, Strengths and Difficulties Questionnaire, Hospital Anxiety and Depression Scale). We used qualitative methods (semi-structured interviews and qualitative data analysis) to describe siblings’ views on illness impact on their lives. Results: Out of 71 eligible families, 38 took part in the study; 29/38 of patients with DMD were wheelchair-dependent. Non-participant siblings were significantly more likely to be females and tended to be younger than the affected child; some families gave current family stress as a reason for not taking part. Participating siblings (n=45) were a mean age of 14 years 6 months (SD 2y 4mo), 22 were female and 23 male; 36 were Caucasian, 23 came from intact homes, and all but six had made adequate or good educational progress. Their mean scores (and rates above cut-offs for problems) on the physical and mental health questionnaires were within expected general population rates. Qualitative interviews identified considerable personal involvement with the affected sibling. Nevertheless, siblings reported little in the way of impact/burden on their lives, whilst 18/38 parents said that the condition had a severe impact on their own lives. Conclusions: Participating siblings of young people with DMD were well adjusted physically and mentally; most impact was reported on main carers. Some non-participating families may be overwhelmed by the condition and/or associated psychosocial problems. Further consideration may be given to ways of accessing and assisting them.

E) National audit of personal practice and experience relating to prevention and treatment of steroid-induced osteoporosis in Duchenne muscular dystrophy

S R CHANDRATRE MRCPCH DNB, R QUINLIVAN FRCPCH FRCP -UK

Objective:To audit personal practice and experience relating to prevention and treatment of steroid-induced osteoporosis in Duchenne muscular dystrophy (DMD). Methods: A questionnaire based on the Consensus Bone Protection Guideline of 2004 was e-mailed to 15 centres managing children with DMD on steroids. Completed questionnaires were returned by e-mail. Data were analyzed anonymously. Results: Eleven of 15 centres treating 315 patients returned the completed questionnaire. Investigations performed before commencing steroid therapy included calcium levels and varicella immune status in 9/11 centres, vitamin D levels in 7/11, and dual energy X-ray absorptiometry (DXA) scans in 6/11 centres, while 9/11 centres had access to DXA scan and facilities for interpretation. Frequency of DXA scans to monitor steroid therapy ranged from 1 to 2 years. Other investigations included spinal X-ray, annual bone profile and vitamin D levels, and vertebral morphometry. Losses of vertebral height or vertebral fractures were recorded in 5.6% of patients with lower limb fractures in 4.7%. Vitamin D or calcium were not routinely supplemented. Dieticians were involved in 8/11 centres. Bisphosphonates could be used in most centres (10/11). Overt vertebral fracture with or without back pain was the consensus indication for treatment with bisphosphonates. Opinion was divided over treatment of reduced vertebral height or back pain, but not overt fracture and long bone fractures. None of the centres recommended prophylactic treatment. Bisphosphonates were used in 28 (8.9%) patients. Pamidronate was used in 6/11 centres compared with risedronate in 2/11 centres. Adverse events reported were mild with good compliance. Some noted dramatically reduced bone fracture pain after infusion. Steroids were continued despite vertebral fractures in other patients. Conclusions: Adherence to the Consensus Bone Protection Guideline 2004 is good but can be improved. Bisphosphonate treatment is safe and well tolerated and needs a consensus guideline on indications for its use.

F) Correlation of hand-held myometry with various methods of assessment of muscle strength and function in patients with Duchenne muscular dystrophy

C MARCHESI MDA,B, M KINALI MD MRCPCHA, M MAIN MA MCSPC, F MUNTONI FRCPCH FMEDSCA - UK

G) Dystrophin positive revertant fibres do not increase with age in Duchenne muscular dystrophy

V ARECHAVALA-GOMEZA MPHARM MSC PHDA, M KINALI MD MRCPCHA, L FENG PHDA, M GUGLIERIB, G EDGE MD PHDC, M MAIN MA MCSPD, D HUNT FRCSE, J LEHOVSKY FRCSF, V STRAUB MD PHDB, K BUSHBY MB CHB MSC MD FRCPB, C SEWRY PHDA, J MORGAN PHDA, F MUNTONI FRCPCH FMEDSCA - UK

Objective: Muscle biopsies from patients with Duchenne muscular dystrophy (DMD) typically show absence of dystrophin.However, dystrophin traces or isolated positive (revertant) fibres are still found in up to 50% of DMD diagnostic biopsies. This phenomenon has been studied in the mdx mouse model for DMD, where revertants increase with advancing age. A number of experimental therapies currently under evaluation are aimed at restoring dystrophin expression in DMD. As dystrophin expression is an endpoint of these early studies, it is important to know if dystrophin expression also accumulates with age in males with DMD.Methods: We reviewed 63 DMD diagnostic muscle biopsy reports issued at the Dubowitz Neuromuscular Unit over the past 8 years to confirm the frequency of revertants and/or traces in the quadriceps muscle. Seven patients, in whom a second biopsy was obtained from the extensor digitorum brevis (EDB) muscle several years after the original diagnostic quadriceps biopsy, were studied to characterize the evolution of dystrophin expression with age. In these patients we performed a detailed comparison of dystrophin expression in the original and the recent biopsies. Results: Revertant fibres were present in 47% of the reports, traces in 33%; in 15% revertants and traces coexisted. In the seven patients who had second muscle biopsies, there was complete concordance between the original and the recent biopsies: presence or absence of revertants was maintained and did not increase with patient’s age. Conclusions: Revertant fibres do not increase with age in males with DMD in the EDB muscle, contrary to what has been reported on mdx mice, where regeneration continues over time. We devised a comparative assay to objectively measure dystrophin expression. Work is in progress to relate the presence of revertants or traces with functional abilities and dystrophin transcription in these patients. Our findings may have important implications for the planning of future dystrophin restoration studies