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