Muscular Dystrophy New Opportunities For Diagnosis and Treatment

Journal of Child Neurology
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Muscular Dystrophy: New Opportunities for Diagnosis and Treatment

Michael A. Babcock, Felina V. Kostova, Richard T. Moxley III, Jeffrey S. Chamberlain and Bernard L. Maria J Child Neurol 2010 25: 1080 originally published online 17 June 2010 DOI: 10.1177/0883073810371000 The online version of this article can be found at: http://jcn.sagepub.com/content/25/9/1080.citation
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Special Issue Article

Journal of Child Neurology 25(9) 1080-1097 The Author(s) 2010 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0883073810371000 http://jcn.sagepub.com
Muscular Dystrophy: New Opportunities for Diagnosis and Treatment

Michael A. Babcock, BS1, Felina V. Kostova, BS1, Richard T. Moxley, III, MD2, Jeffrey S. Chamberlain, PhD3, and Bernard L. Maria, MD, MBA4
Muscular dystrophies are characterized by progressive muscle weakness. The most common form, Duchenne muscular dystrophy, will affect more than 400 boys born in the United States within the next year. Since the discovery of dystrophin in the late 1980s, research has focused on treatments at the molecular level. This is an exciting time in the field of muscular dystrophy, as many of these treatments are approaching clinical trials. Recent advances in care have also led to the development of standardized care guidelines. With this backdrop, the 2009 Neurobiology of Disease in Children Symposium, held in conjunction with the 38th Annual Meeting of the Child Neurology Society, aimed to: (1) describe current clinical management, (2) review recent advances in understanding of pathogenesis, (3) discuss therapies and clinical trials, and (4) define future research directions. This article summarizes the presentations and includes an edited transcript of question-and-answer sessions.
CLINICAL ISSUES
Moderator: Richard T. Moxley, III, MD, University of Rochester, Rochester, New York
Muscular Dystrophy: Where Are We Now? Francesco Muntoni, MD, UCL Institute of Child Health and Great Ormond Street Hospital for Children, London, United Kingdom
Dr Muntoni discussed concepts in the field of muscular dystrophy. The first is that although there is a wide clinical diversity in muscular dystrophy, relatively few pathogenic mechanisms are responsible for muscle degeneration. As these mechanisms are more fully understood, a purely clinical basis of classification is no longer sufficient. These mechanisms create relationships between previously separate muscular dystrophies, which can now be grouped according to the class of protein affected by each. In this classification, muscular dystrophies can be separated into those that affect the transarcolemma, extracellular matrix, enzymes, nuclear envelope, sarcomere, or others. For example, dystrophin and sarcoglycan are both proteins
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associated with the sarcolemma. It should not be surprising that deficiency in each of these proteins results in a Duchenne-like clinical picture. Dr Muntoni used proteins involved in the extracellular matrix to more fully explain this concept. A number of extracellular matrix proteins, and cell surface proteins that bind extracellular matrix proteins, when deficient, cause congenital muscular dystrophies. For example, collagen VI deficiency can lead to Ullrich congenital muscular dystrophy, characterized by a combination of distal laxity, proximal contractures, early rigidity of the spine, and respiratory failure, with only mildly elevated serum creatine kinase levels. Ullrich congenital muscular dystrophy results from recessive or de novo dominant mutations of the collagen VI genes. Within the past decade, it was discovered that Bethlem myopathy also results from deficiency in collagen VI, but is due to dominant mutations of the collagen VI genes. Bethlem myopathy, a much milder form of congenital muscular dystrophy, is characterized initially by laxity and mild proximal weakness in infancy. There is then progressive flexion contractures of the elbow, interphalangeal joints, and Achilles tendon, and rigidity of the spine. As in Ullrich congenital muscular dystrophy, serum creatine kinase levels are only mildly elevated. So, deficiency in collagen VI can result in both the more severe Ullrich congenital muscular dystrophy and the less severe Bethlem myopathy, depending on the mutations involved. This highlights the second concept that there is a continuum between the congenital forms of muscular dystrophy and the milder, adult-onset forms.
College of Medicine, Medical University of South Carolina, Charleston, South Carolina 2 Department of Neurology, University of Rochester Medical Center, Rochester, New York 3 Department of Neurology, University of Washington School of Medicine, Seattle, Washington 4 Department of Pediatrics, Medical College of Georgia, Augusta, Georgia Corresponding Author: Bernard L. Maria, MD, MBA, Medical College of Georgia, 1446 Harper Street, BT-1850, Augusta, GA 30912-3700 Email: bmaria@mcg.edu
Babcock et al Other extracellular matrix protein deficiencies can lead to a similar clinical picture. An Ullrich-like form of congenital muscular dystrophy characterized by distal laxity, rigidity of the spine, and less severe respiratory failure with normal collagen VI expression was recently described. It was found to be caused by a mutation in the integrin alpha 9 gene. Therefore, a mutation in a different protein affecting the extracellular matrix produces a clinical picture similar to collagen VI deficiency with congenital onset of an Ullrich-like syndrome. The pathogenic mechanism by which deficiency in collagen VI results in muscular dystrophy is beginning to be more fully understood. Deficiency of collagen VI introduces a number of secondary events in the muscle fibers, which eventually induce a dysfunction of the mitochondria through abnormal activation of the permeability transition pore. This leads to muscle fiber apoptosis and silent muscle loss with normal serum creatine kinase. These secondary changes can be targeted for therapy. Cyclosporine is able to inhibit the opening of the permeability transition pore, and both animal work and a small pilot study in patients with collagen VI deficiency have shown a decrease in apoptosis after treatment with cyclosporine. Unfortunately, cyclosporine also has immunosuppressive activity. This led to the development of a new drug, Debio 025 (Debiopharm, Lausanne, Switzerland), which selectively inhibits the permeability transition pore without affecting the calcineurin immune response pathway. Administration of Debio 025 was very effective in animal models of collagen VI deficiency and this interesting candidate could be taken into clinical trials for collagen VI deficiency. Within the past decade, enzyme deficiencies have been linked to muscular dystrophies. In particular, mutations in the genes encoding enzymes involved in the glycosylation of dystroglycan are responsible for the dystroglycanopathies. The mechanism of dystroglycan glycosylation is uncommon, requiring O-mannosylation, and a number of the genes responsible for the dystroglycanopathies are involved in this step. The others, through unknown mechanisms, all lead to the stripping of sugars from dystroglycan. Binding of dystroglycan to laminin is mediated by sugar, and the absence or reduction of these sugars results in a reduced or abolished capacity for dystroglycan to bind laminin. Each of the gene defects in this category were originally assigned to an individual condition, with fukutin deficiency in Fukayama muscular dystrophy being a typical example. However, all of the proteins in this class can cause a phenotype of variable severity depending on the percentage of reduction in glycosylation. This leads to a clinical continuum ranging from limb girdle muscular dystrophy 2I with proximal weakness and normal brain structure to Fukayama muscular dystrophy with congenital onset and pachygyria, to the most severe end of the spectrum, WalkerWarburg syndrome with congenital onset and lissencephaly. In each of these conditions along the spectrum, serum creatine kinase is markedly elevated. As stated before, it was thought that each of these conditions was because of a particular gene defect, with limb girdle muscular dystrophy 2I because of mutations in the fukutin-related protein gene. However,
1081 patients with Walker-Warburg have been identified with mutations in the fukutin-related protein gene. Furthermore, while mutations in fukutin give rise to Fukayama muscular dystrophy in Japan, mutations in this same gene can give rise to anything along the spectrum from severe Walker-Warburg syndrome to mild limb girdle muscular dystrophy if it occurs elsewhere. Therefore, it is not the gene defect itself that causes a particular phenotype, but the effect of the mutation on the glycosylation of dystroglycan. As a final example, deficiency in the nuclear envelope proteins emerin, laminin, and nesprin all result in an EmeryDreifuss muscular dystrophy phenotype. The clinical features of Emery-Dreifuss muscular dystrophy are early contractures of the Achilles tendons, elbows, and spine, slowly progressive muscle wasting and weakness with a predominately humeral and peroneal symmetric distribution, invariable cardiac conduction defects, and serum creatine kinase elevated to a lesser extent than in Duchenne muscular dystrophy. X-linked EmeryDreifuss muscular dystrophy, because of mutations in emerin, always presents clinically as the same condition. However, the laminopathies, which are much more common, have a diverse spectrum of clinical severity. Nesprin is the most recent nuclear envelope protein found to cause an Emery-Dreifuss-like condition. More examples of new variants will continue to come forward and clinicians must watch out for these in clinics. Dr Muntoni closed by reminding the audience that many genetically undefined forms of muscular dystrophy remain and need to be explored. Also, this is a unique time to build on the example from the Duchenne muscular dystrophy field and compile the natural history studies of other muscular dystrophies.
The Molecular Basis for the Childhood Muscular Dystrophies Katherine Wagner, MD, PhD, The Johns Hopkins Hospital, Baltimore, Maryland
Dr Wagner discussed 3 examples of discoveries over the past 2 decades in the molecular mechanisms underlying some of the childhood muscular dystrophies and their impact on advances in therapeutics. The discovery of the Duchenne gene and the identification of dystrophin were pivotal in unlocking the pathophysiology behind muscular dystrophies. This led to the purification of a complex of tightly associated and highly glycosylated proteins, the dystrophin-glycoprotein complex. Perturbations in these proteins, or in their glycosylation, can lead to disease. When the protein loses its glycosylation, it loses the epitope that binds to the extracellular matrix. This is seen in congenital muscular dystrophy 1C, which is because of mutations in the fukutin-related protein (FKRP) gene that glycosylates alpha-dystroglycan. Other breakdowns in the structural support of muscle cells can also lead to disease, including loss of the link with the basal lamina because of loss of collagen VI in Ullrich muscular dystrophy and loss of laminin 2 in merosin-deficient muscular dystrophy. The
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1082 basic understanding of the importance of this structural link between the cell and the extracellular matrix allows pursuit of therapies aimed at increasing the structural integrity of this link, and alternative protein-protein interactions that may strengthen this connection are being explored. Studies into the pathogenesis of myotonic muscular dystrophy have shown how RNA processing may cause disease. Myotonic dystrophy type 1 is caused by expansion of CTG repeats in the 30 untranslated region of the DMPK (myotonic dystrophy protein kinase) gene on chromosome 19. Patients display facial and distal limb muscle wasting and weakness and the characteristic electrical hyperexcitability of muscle, which the patient perceives as an inability to relax a muscle. This disorder is autosomal dominant and exhibits extreme anticipation, with children manifesting disease approximately 29 years earlier than parents. Unaffected individuals have 5 to 36 CTG repeats, while those with the classic mutation have 100 to 1000, and those with congenital onset have more than 1000. The first clue to the molecular basis of this disease came from the observation of ribonuclear inclusions, which suggested the disease was RNA-mediated. A protein that bound trinucleotide repeats of CUG was found. An increase in this CUG binding protein leads to alternative splicing of the skeletal muscle-specific chloride channel mRNA and the insulin receptor mRNA, resulting in the myotonia and insulin resistance characteristic of myotonic dystrophy. Another protein, muscleblind-like protein, binds to the hairpin loop of DMPK mRNA created by the CUG repeats. Muscleblind-like protein affects alternative splicing by antagonizing the effect of the CUG binding protein. However, when muscleblind-like protein binds to the CUG repeats in DMPK mRNA, it is sequestered in the nucleus and is no longer able to antagonize CUG binding protein. While the mechanisms and interactions between CUG binding protein, muscleblind-like protein, and the CUG repeats in DMPK are not fully understood, this basic knowledge opens the door to potential treatments, including those that would increase muscleblind-like protein, decrease CUG binding protein, or mask the hairpin loop created by the trinucleotide repeats. Interestingly, congenital myotonic dystrophy may not have the exact same disease mechanism as adult-onset myotonic dystrophy. The allele containing the CTG repeats is hypermethylated in congenital myotonic dystrophy, and this may alter expression of nearby genes. Congenital myotonic dystrophy is marked by abnormal prenatal development with decreased fetal movement and polyhydramnios. If patients survive the perinatal period, they stabilize and develop features of adult-onset myotonic dystrophy. Therefore, the congenital features may be an epigenetic phenomenon compared to the RNAmediated process seen in adult-onset myotonic dystrophy. This leads to the third example. Facioscapulohumeral muscular dystrophy appears to be an epigenetic disorder. It is autosomal dominant and is caused by a contraction of subtelomeric macrosatellites, D4Z4 repeats, on chromosome 4q. Normally, there are between 11 to 100 repeats. However, patients with facioscapulohumeral muscular dystrophy carry alleles with
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Journal of Child Neurology 25(9) only 1 to 10 of these units. The D4Z4 repeat region is highly methylated, with features of heterochromatin. An attractive model for the pathophysiology of this disease process is that a contraction in the number of repeats inhibits binding of D4Z4-repressing complexes, leading to a greater transcription of neighboring genes. Unfortunately, an increase in neighboring proteins has not been seen in biopsies of affected muscles. An alternative hypothesis points toward DUX4, an open reading frame within the D4Z4 repeats with many processed and alternatively translated transcripts, all of which are new candidates for playing a regulatory role in facioscapulohumeral muscular dystrophy, and may be future therapeutic targets. This lack of molecular understanding of facioscapulohumeral muscular dystrophy led to the creation of a Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center for Facioscapulohumeral Muscular Dystrophy Research. One of the early goals of this collaboration is to increase the quantity of biomaterials, animal models, and cell lines. The collaborative is biopsying patients and unaffected first-degree relatives. In the first year, 6 cohorts have been collected, and it is on track to recruit 30 cohorts over the next 5 years. Dr Wagner closed by briefly presenting some preliminary results from these biopsy specimens. Interestingly, cells from severely affected muscles and unaffected muscles of the same patient behave differently in vitro. In looking at some candidate genes, FGR2 appears elevated, while FGR1, a gene whose elevation replicates the disease in a mouse model, is unchanged, and ANT1 shows decreased expression.
Duchenne Dystrophy, the Diagnostic Odyssey, and the Centers for Disease Control and Prevention MD STARnet Katherine D. Mathews, MD, University of Iowa Childrens Hospital, Iowa City, Iowa
Dr Mathews introduced the concept of MD STARnet, which stands for Muscular Dystrophy Surveillance Tracking and Research Network. MD STARnet is a Centers for Disease Control and Prevention (CDC) cooperative agreement with Arizona, Colorado, Iowa, western New York, and, more recently, Georgia and Hawaii. It was created with the goals of describing and defining the Duchenne/Becker muscular dystrophy population, defining and describing health-care access and outcomes for patients, and, importantly, distributing this information to guide care and policy. The goal of this population-based surveillance study is to indentify every patient with Duchenne/Becker dystrophy born after 1982 and residing in these geographic locations. The concept is to identify every potential case, abstract information from the medical record, send selected data to a clinical review committee for diagnostic assignment, and finally input abstracted data into a pooled database available for analysis. The surveillance data are gathered retrospectively, and the quality of the data is dependent on how accurate the diagnoses are. Each case gets a specific diagnostic assignment of
Babcock et al definite, probable, possible, asymptomatic, or affected female. A definite case has symptoms plus either a documented dystrophin mutation, a diagnostic biopsy, or elevated creatine kinase with X-linked family history and an affected family member with documented dystrophin mutation or diagnostic biopsy. A probable case fails to meet the rigorous standard of a definite case, but the vast majority of these cases have Duchenne/Becker muscular dystrophy. These 2 categories are usually included in analyses. Possible cases have too limited information. However, information is abstracted every year, so these cases are reconsidered annually. The other diagnostic groups are not considered in data analysis. With this criteria, as of June 2009, there were 573 definites and 61 probables available for analysis. There are obvious advantages to doing a population-based epidemiologic study. It is not affected by any 1 doctors patient-cohort or style and is not influenced by voluntary participation bias. The limitations include the constraints of retrospective analysis of abstracted data that is dependent on the medical record. Prevalence data of Duchenne/Becker muscular dystrophy from MD STARnet ranges from 1.25 to 1.78 per 10 000 males age 5 to 24 years. This is consistent with previous estimates and indicates reasonable ascertainment. For cases where the mutation is known, there is a similar distribution of mutation type, which indicates the population is fairly representative. There are many works in progress that use the collected data. There are articles on steroid usage, fractures, mutation analysis, cardiomyopathy, respiratory insufficiency, and mental health issues that will be published soon. Dr Emma Ciafaloni from the University of Rochester recently published a paper documenting the diagnostic delay in Duchenne muscular dystrophy. The purpose of this study was to determine the time course from first symptom onset to diagnosis. Retrospectively, parents first recognized the earliest signs and symptoms around age 2.5, and first mentioned these to a health care provider at age 3.5. Children first saw a neurologist and were tested for creatine kinase levels at 4.5, and the diagnosis was made close to age 5. Overall, there is a 2.5-year delay in diagnosis. There was also a difference in early signs by age group. While gross motor delay, weakness, and trouble walking were all common symptoms, 16% of patients under age 1.5 presented with failure to thrive, 18% of patients 1.5 to 3 years of age presented with speech delay or articulation problems, and approximately 25% of patients 3 to 5 years of age had cognitive or behavioral issues. While most pediatric neurologists know the constellation of these early signs and symptoms, those that first identify the signs and symptoms do not, and these first identifiers must be reached out to and educated. Parents and school personnel first identify the signs and symptoms in 75% of patients by age 2.5. This is the most difficult group to reach, but it is possible. Families need to be educated to express their concerns with primary care physicians earlier. If the school expresses a concern, families should be encouraged to consult the doctor soon. Once parents do consult a physician, 64% of the time a pediatrician or family practitioner is the first person notified,
1083 compared with 9% for neurologists. Only 35% of primary care providers ordered testing of creatine kinase levels in this setting, while only an additional 17% referred the patient to a neurologist, who then ordered testing of creatine kinase. Sixteen percent referred the patient to a developmental occupational or speech therapist and 5% referred only to an early access developmental stimulation program. While it may be difficult to change anything at the parental level, it should certainly be possible to educate our primary care colleagues. Early diagnosis is important for family peace of mind, genetic counseling, getting a diagnosis, early management, education, physical therapies, and starting steroids. The field is quickly reaching a point where making a genetic diagnosis will affect how a child is treated. The earlier the treatment is started, the better prognosis a patient will have. To this end, the CDC has funded a number of initiatives to improve early diagnosis. The National Task Force for Early Identification of Childhood Neuromuscular Disorders is a consortium of agencies whose concept is to get all partner agencies to assist in getting this message out. There are 3 key messages: (1) recognize motor delay, (2) initiate diagnostic testing of creatine kinase, and (3) seek early diagnosis. Many primary care physicians never see their patients walk, and they should be encouraged to watch children walk and run. Creatine kinase is a cheap, easy test and if there is any concern about the diagnosis, physicians should consider a creatine kinase. Referral to early access is not enough. Lastly, early diagnosis is important. The field has reached a point where there are therapies that can make a difference in a childs life, and these may be most beneficial if started early. The plan to disseminate this message is outreach to educators across disciplines. One part of this is the development of educational videos that physicians can download from the Internet to assist with early recognition and to use in lectures. Despite dramatic improvement in diagnostic capability, the 2.5-year delay in diagnosis is comparable to historical reports. Decreases in diagnostic delay will come from education of other care providers, not technological advances.
Duchenne Muscular Dystrophy: TREATNMD and Centers for Disease Control and Prevention Guidelines for Care Katherine Bushby, MD, University of Newcastle upon Tyne, Newcastle, United Kingdom
Dr Bushby presented a collection of new guidelines for the diagnosis and management of Duchenne muscular dystrophy. These guidelines were developed as part of an international collaboration between the CDC and the TREAT-NMD European Network, with input from international patient organizations. These guidelines appeared in the January and February 2010 issues of Lancet Neurology. Importantly, care standards are only as good as their dissemination and implementation, and Dr Bushby urged everyone to assist in their distribution. The care standards come at an exciting time, as many new treatments are coming into the clinical arena. These include
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1084 exon skipping, stop codon mutation suppression, gene transfer, cell therapies, and others. As new trials in large multicenter studies are introduced, it is imperative there exists a standard level of care so trial data can be meaningfully interpreted. Also, patients deserve harmonization of care. Studies have shown that improved management in Duchenne muscular dystrophy is improving survival. The introduction of novel treatments such as home nocturnal ventilation and spinal surgeries are now pushing survival into the 30s and beyond. Patients should have equal access to different therapy modalities. A standardized set of recommendations provides a tool to lobby health-care providers to raise standards across the board. The best way to gather evidence for development of standards of care is through meta-analyses of high-quality randomized control trials. For some areas of Duchenne muscular dystrophy management, such as corticosteroid use, these are available. However, randomized control trials in many areas of care are lacking, and so expert panels, representative of the best levels of care across different disciplines, have been assembled. The RAND/UCLA Appropriateness Method was chosen as the unbiased method to build consensus and generate recommendations. First, key areas of clinical management were identified in 8 disciplines. Under the broader heading of neuromuscular and skeletal management fall the disciplines of diagnostics, rehabilitation management, orthopedic management, and corticosteroid management. Management of other disciplines includes gastrointestinal (which included speech/ swallowing and nutrition), cardiac, pulmonary, and psychosocial management. A panel was formed for each discipline to generate recommendations. For each discipline, the personnel and toolkit required for optimal care were defined. The consensus generation was geared toward a pragmatic, clinically relevant set of directions for care in specific scenarios, with an emphasis on multidisciplinary involvement and coordination of care. The chosen model places patients and their families, along with their care coordinator, at the center of the management program. Management in Duchenne muscular dystrophy changes as the disease progresses, and different disciplines must be brought in and out. Five discrete stages of disease progression were identified: presymptomatic, early ambulatory, late ambulatory, early nonambulatory, and late nonambulatory. All of the different tools and interventions necessary for each discipline were mapped to the different stages. Diagnostics is biased toward the early stages, where anticipatory planning is also very important. Knowing the progression of Duchenne muscular dystrophy allows the care-coordinating physician to anticipate future problems rather than wait for them to arise. One key theme, which recurred in several areas, was the necessity to have a good set of functional assessments applied rigorously in patients. Another key theme was management with an eye toward the care of adult patients. Concerning the diagnostic stage, for children with no family history of Duchenne, not walking by 16 or 18 months or having a Gowers sign at any age should trigger a diagnostic work-up, as should any suspicion of abnormal muscle function in
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Journal of Child Neurology 25(9) children with a family history. Additionally, unexplained elevation of transaminases is a factor that needs to be taken into greater consideration. While electromyography may not be necessary in straightforward cases, precise molecular testing is always necessary, as it not only allows for genetic counseling, but also as mutation-specific therapies are developed, it allows physicians to appropriately tailor treatment. The key message from the neuromuscular group was that corticosteroid use aims to maintain muscle strength and function as much as possible to allow integration into the community and participation in activities. The most appropriate time to initiate corticosteroid treatment, and also monitor response to treatment, is the plateau phase, which can only be determined with the use of systematic measurements of function and strength. This must be combined with systematic prophylaxis and management of the predictable side effects of steroids. The rehabilitation group sought to correct the lack of pragmatic guidance for physiotherapy regimens and laid out key recommendations about the combined use of home stretching, clinical stretching, and monitoring. This ties in closely with orthopedic input, and the orthopedic panel made recommendations concerning management of contractures and scoliosis. A particular concern addressed the timely and appropriate provision of wheelchairs and seating with an emphasis on maintaining functionality and allowing integration as much as possible rather than focusing simply on disease management. The respiratory care and cardiac care panels looked at management of complications, as respiratory failure, cardiac failure, or a combination of the 2 is what usually leads to death. Furthermore, general principles of management in these 2 areas have been shown to improve quality of life and survival. One of the key messages is prophylaxis and acquiring baselines to assist with monitoring. For respiratory management, this includes assessing peak performance in different areas early on and prophylaxis with immunizations, prompt treatment of chest infections, and careful perioperative care. A properly planned prophylactic management strategy allows many of the great emergencies and hospital admissions to be avoided. Planning surgery and anesthesia is an important issue, as this is often when patients encounter problems. Clear guidelines are given about pre and postsurgical monitoring. Nocturnal ventilation may be required prior to surgery in boys who are at a critical level of respiratory function. Dr Bushby and colleagues analyzed the Duchenne muscular dystrophy population in Newcastle prior to systematic cardiac screening and found that patients who clearly died of cardiomyopathy tended to be younger than those who died of respiratory complications. It is difficult to recognize progressive fall in left ventricular function in patients restricted to wheelchairs and subjected to minimal stress, as the onset of heart failure symptoms are a very late phenomenon. Intervening before symptom onset, while left ventricle function is still within the normal range, is more likely to bring benefit, and current trials are looking at the effect of intervening at different times, including at diagnosis. A cardiologist should be involved in all stages of the disease, and cardiac investigations,
Babcock et al including echocardiography, should be done at diagnosis to establish a baseline. These should then take place every 2 years and prior to surgery until age 10, then annually. Treatment with angiotensin-converting enzyme inhibitors, and beta-blockers, if tolerated, are first-line therapies initiated with any change in baseline. The gastroenterology group looked at the risk of patients becoming overweight or underweight. Patients on steroids have an additional risk of becoming overweight and this needs to be actively managed. Assessments by speech and language therapists are appropriate at different stages, particularly in the later stages when patients may be unable to feed easily. Gastrostomy and nasogastric feedings should be considered earlier to enable a good standard of living, rather than left to the end stages as rescue medical management. One issue that has not always received appropriate consideration is psychosocial care. This panel developed pragmatic and useful recommendations that should raise awareness of the types of interventions available for patients. Patients with Duchenne muscular dystrophy are at risk for problems with learning and behavior, and recognition of problems at an early age is the key to successful intervention. This is important on the learning side, where recognition of specific learning patterns can help lead to a personalized educational plan for a child, and on the behavioral side with regard to coping and adaptation in the family. Both counseling and drug administration are well-documented interventions that should be used. Support for the family must be emphasized by the primary care-coordinator. The introduction to patient groups is both valuable and necessary. As the medical care for these patients progress, more and more children are living into adulthood. Schools and social services lag behind and are not gearing children with Duchenne muscular dystrophy toward active participation in adult life. Dr Bushby closed with a plea to physicians to educate these organizations and help the social care for these patients progress. These organizations must be engaged in being more proactive in their approach to patients and planning for the emerging adult generation of patients.
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The first question, clearly the child who has motor delay out of proportion to their cognitive delay should be the red flag. I went through a bunch of signs and symptoms in my talk. I do CKs [creatine kinases] pretty liberally. My attitude is if you dont have an underlying explanation for the developmental delay and motor involvement is part of it, do a CK [creatine kinase] as part of your workup. Audience Member: Dr Mathews, do you have the information as to what percent of children who turn out to have Duchenne walked independently by age 1? Dr Mathews: We would have that. I dont have that information off the top of my head. I know average walking age is around 16 months. Audience Member: Every year for the last decade I have seen 1 and usually 2 children who have gone so far as to have a liver biopsy because theyre not weak and they have a liver enzyme elevation they got during some intercurrent illness. Im sure thats not a unique experience. That wouldnt show up in your diagnostic delay paper, but how often does that occur? Dr Mathews: We do have data about that, again I cant give you the numbers, but of the whole group its probably less than 1%. But I think everybody who manages these diseases has this issue. I had 1 patient 2 weeks ago who came in with elevated transaminases that fortunately did not go as far as liver biopsy. But elevated transaminases is one of the routes that was not high frequency enough to make that list. But its definitely another group in which we need to be talking to our colleagues in GE [gastroenterology]. Audience Member: I want to make a comment about the question concerning newborn screening that would address potential undiagnosed patients. From the cases in your study, there were at least the 2 groups that have been doing newborn screening CK [creatine kinases]. I think Ann Marie was doing a pilot project, and also probably Ohio State. There were enough data and theyre actually quite sensitive picking up, just by doing spot CK [creatine kinase], the kids with Duchenne. They are not in the 10 000 range, but its clearly higher than the normal range. Dr Mendell: The ones weve identified have CKs [creatine kinases] over 2500. We took a cutoff at 600 for dry blood spots and then have analyzed all DNA samples over 600 on dry blood spots, and the 2 that were positive so far were over 2500. Dr Landis: So one of the things that National Institutes of Health (NIH) and the American public are coping with is health-care reform. A piece of that is when best practices are identified. How do we track their acceptance into the clinical community, and Im wondering how you all plan to do that? Is there the possibility of experiments using different strategies that would enable us to know better? We all know that we should exercise and lose weight and we dont do that. I imagine these guidelines will actually be more successful because there are kids involved, but I would be interested in your thoughts of how we can take best advantage of this effort to look at how things actually get implemented, not just disseminated. Dr Bushby: I think MD STARnet is an excellent way to make a start on that because you will have the baseline data of whats going on. You can then take this out and disseminate 1085
Questions and Answers

Dr Maria: I have a couple of questions on the issue of the challenge of early diagnosis. What are your thoughts in terms of the primary care pediatrician and specific approaches or cues, and short of doing a CK [creatine kinase], what do you consider some of the hallmark red flags? The second question, is there any data in terms of perinatal or prenatal history or evaluation that might allow for an even earlier suspicion of involvement that might then allow for an earlier diagnosis? Dr Mathews: Ill address the second question first. These kids look normal at both, and I dont think there is anything such as ultrasound that would work. Aside from doing a prenatal CK [creatine kinase] or something like that, I dont think there are any prenatal clues. Parents dont come in and complain of decreased fetal activity or any of those kinds of things.
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it across those states and youll have an answer in a couple of years as to where practice mightve changed and where youve been able to make a difference. You will already have the baseline from that. Thats part of, as I understand it, the same investment that the US government made into MD care. MD STARnet and the CDC Care Considerations were all part of the same package. So, theres a perfect way to do that. When we all think about making these educational videos, it doesnt only need to relate to diagnosis. We could extend that educational program to all of care and we could then monitor the use of these and how these are applied in different areas. In Europe, weve gotten more EU [European Union] funding for a project called Care and MD, which will set up training programs for the Eastern European countries and which has built into it both the evaluation from the patients and clinicians perspectives through the Care and Trial Site registries of how they are applying these different standards. I think we have the tools at our disposal to do this properly. Now we need to think about how we apply this. The problem, if there was any problem with this process, is that it was such a process and we really had to work to make something meaningful and clinically relevant out of a huge mass of 70 000 different clinical scenarios. Because it has been so process driven, this has taken up everybodys thinking time. Actually, we have always had an eye on dissemination and implementation, and Id like to talk to those of you who are involved in these projects to see how we can really push that forward. And I know weve got the support of MDA [Muscular Dystrophy Association] and of PPMD [Parent Project Muscular Dystrophy] and of the different patient organizations in doing this too. Audience Member: The TREAT-NMD website has been a wonderful resource for us all. I have a question regarding the outcome measures. I was trying to figure out whether 1 or 2 can be used for part of my study or as clinical care. I was completely overwhelmed. There are more than 50. Is there any effort to try to streamline this and make core recommendations, or outcome measures, so people can adopt these in their study or practice, or different studies can be compared easily? Dr Bushby: Absolutely. Thank you for your comment about the Web site. You will find on the Web site, a registry of outcome measures, which has a lot of detail about different outcome measures that can be used in different diseases. Those of you who know of outcome measures that are useful for muscle diseases, please contribute to the registry of outcome measures because that is the way it is going to get better and more widely used. Please see me if you want more information about that. In terms of recommendations for particular studies, weve so far focused on trying to understand whats out there. We have a meeting being planned for next year where we will take all of the natural history data generated using different outcome measures and compare it side by side for factors such as reliability, sensitivity to change, and so forth. In terms of choosing an outcome measure, it depends on what you want to use it for. That was 1 of the key things we learned over this period; for a particular disease there is not just 1 good outcome measure. There may be a range of good outcome measures depending on the stage of the disease and what youre trying to do. For example, PTC looked at all of the outcome measures that we 1086
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were using for years and wouldnt allow the use of any of them in our study. They went for the 6-minute walk test because that had prior regulatory experience and had been used before for regulatory approval. So there will always be considerations, which are different depending on your study. For clinical practice, in the care guidelines theres a list of good, well-used clinical scales. Really, the recommendation is to find one that your physiotherapist and your clinic can be familiar with and apply it regularly. With any of these scales, the Brooke scale, the North Star, the Hammersmith, and others, you will get very clear data across your population that you can use clinically, and these are the kind of things that we also want to apply into the studies. Dr Maria: I want to thank you for the incredible heavy lifting thats involved in pulling this together in a set of guidelines. My question has to do with underserved communities. Given your point about the essentials of the multidisciplinary involvement, or really interdisciplinary care, so many of the patients we serve are in small, underserved communities, so I wonder if your panels addressed how to engage the community broadly at churches, community groups, above and beyond the primary care physician, to ensure some compliance, particularly in patients who have to travel significant distances to core centers. Dr Bushby: Yes. We were very aware of the point that Dr Mathews made that those who participate are not necessarily the ones that we need to be most worried about. Its the ones who we never hear about, the people who are just captured as part of your data collection that sit and take whats given to them and never realize that theres anything better out there. So, I think this has to be part of the outreach and has to be part of the communication to pediatricians. I think we, the pediatric neurology audience, should already be well aware of all of these findings and implications and applications. Taking this out that step further to the people who dont have access to a neuromuscular pediatrician is important. Its also up to us to promote the establishment of good clinics in underserved areas so that what is now an underserved area may, in a few years time through strategic planning of the MDA or of the other organizations, actually become a place where somebody does start to run a good clinic and does start to offer those services to the patients. But it isnt going to happen overnight, particularly in a huge country like the United States. Dr Cwik: At MDA, weve been thinking about this issue as well because there are huge parts of the country where we dont have MDA clinics. Trying to put a clinic in Wyoming or the middle of Montana is difficult. Weve got some there, but there are still big expanses of land where people dont have access to a multidisciplinary clinic. Initially, weve been thinking about how to get care to those kinds of areas. Even in some of the more populated areas we have a real shortage of experts in neuromuscular diseases. We are looking at ways to think outside of the box to get the information out there, with things like outreach meetings, having our core centers, inviting participation from physicians from under-served communities, telemedicine, webinars, and other kinds of new technologies to start spreading the message in a better way. Dr Bushby: I think networks are very important. You can have a center that has outreach, and that center serves a network of local clinicians who only see 1 or 2 cases themselves but
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have access to the resources of the whole network when theyre looking after their patients. That models worked very well in Scotland to train people in a very geographically dispersed area.
1087 required nocturnal ventilation, while about half the untreated patients did. Another study focused on cardiac benefits and looked at annual echocardiography studies. In the treated group, 93% of patients had no ventricular dysfunction, compared with 53% in the untreated group. To complete the discussion of long-term effects, Dr Moxley summarized results from his Rochester MD STARnet group. The cohort is a group of 73 patients with Duchenne muscular dystrophy born between 1971 and 1990 who received prednisone for more than 3 years. The mean follow-up period was 14.6 years. Similar to other studies, treatment with corticosteroids increased age of cessation of ambulation, peak forced vital capacity, and age at which peak forced vital capacity occurs. This might increase both quality of life and lifespan. While Dr Moxley and colleagues have not directly studied the survival effects of steroid treatment, the treatment group at Rochester seems to be headed in the direction of increased survival, with 42 of the 73 patients still living, with an age range of 18 to 34 years. Further research is needed, but more patients will soon be getting married, traveling independently, and starting businesses. The side effects of steroid treatment are predictable, as steroids have been studied for a long time. The major side effects that cause problems are weight gain, behavioral problems, cushingoid facies, and decreased linear growth. Of the 73 patients in the Rochester group, 18 patients discontinued therapy, mainly because of weight gain. Next, Dr Moxley briefly covered some management strategies. Duchenne muscular dystrophy progression can be broken down into ambulatory, intermediate, and late stages. Within the ambulatory stage, there are 3 phases. Initially, patients continue to improve in their motor capabilities, then they reach a plateau phase, and finally they decline. The recommendation is to start treatment with prednisone during the plateau phase at 0.75 mg/kg/d. The protocol at Rochester for children in the ambulatory phase includes 3- to 6-month interval monitoring of weight, height, blood pressure, mood, skin, and forced vital capacity. Patients with increasing orthopedic complications have entered the intermediate stage. In addition to continued monitoring, orthopedic, pulmonary, and cardiac colleagues should be included in management to optimize the safety of any operative orthopedic procedure. There are also late-stage complications, but with early monitoring Dr Moxleys group is already seeing less severely affected individuals. This is how the benefits and justifications of early monitoring recommendations in the guidelines should be sold to families. One thing to discuss with families is when to initiate steroid treatment. The CDC guidelines recommend initiation at the plateau phase, but it may be helpful to begin even earlier. One could initiate treatment after a patient has begun walking and has completed immunizations. Importantly, parents must have a good comprehension of the reason behind beginning treatment, and understand the requirements. Patients must have regular monitoring for side effects, including monitoring weight gain, cushingoid facies, behavioral changes, growth
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CORTICOSTEROID TREATMENT
Moderators: Gerald Fenichel, MD, Childrens Hospital at Vanderbilt, Nashville, Tennessee, and Douglas Biggar, MD, University of Toronto, Toronto, Canada
Long-Term Corticosteroid Therapy: Side Effects and Management Richard T. Moxley, III, MD, University of Rochester, Rochester, New York
Dr Moxley discussed the benefits and side effects of corticosteroid therapy, the only known treatment that slows muscle weakness and improves muscle function. As a brief history, steroid therapy has its roots in the Clinical Investigations in Duchenne Dystrophy group, which developed from 1979 through 1993, and unfortunately, became an authority on negative therapeutic trials, with leucine, nifedipine, penicillamine and vitamin E, and mazindol, showing little effect on patients. Finally, the group decided to look at prednisone, and an openlabel study found that compared with the natural history data the group had compiled, average muscle strength scores improved. This led to more trials of prednisone by the group. One study looked at the change in muscle strength score in 34 muscle groups over a 6-month period and found both doses of 1.5 mg/kg/d and 0.75 mg/kg/d of prednisone produced virtually identical improvements by 3 months that were maintained at 6 months. Average muscle strength scores, timed function tests, and vital capacity also significantly improved. A similar study found a 0.3 mg/kg/d dose of prednisone also resulted in significant improvement, but was less effective than the higher doses. Additionally, studies that looked at alternate-day treatment showed a decline in muscle strength that paralleled the natural history. These studies formed the basis for Dr Moxleys practice parameter recommendations in Neurology to start patients on a 0.75 mg/kg/d dose and decrease by 25% increments every 1 to 3 months for side effects. Dr Moxley next reviewed the long-term data on steroid therapy. One study found boys treated with 0.55 mg/kg/d of steroid walked for 3.3 years longer and had less scoliosis than untreated controls. One side effect noted was that 32% of the treated group had vertebral fractures. Though this was by X-ray diagnosis and did not hamper the patients, this complication warrants monitoring. Another study looked at the longterm benefits of deflazacort in patients second decade. Forty patients, treated with a dose of 0.9 mg/kg/d, walked 3 to 5 years longer than controls. Treated patients had significantly less scoliosis and less ventricular ejection fractions less than 45%. Furthermore, none of the treated patients over age of 15 years
1088 retardation, fracture risk, cataracts, and others. All patients need a medical alert card. The response to side effects is important, and parents must be urged not to stop steroid treatment abruptly. The typical reason for reducing dosage is weight gain, and the typical response is to decrease dosage by 25%. While steroid therapy has come a long way, there are many unanswered questions. The cellular mechanisms that account for the beneficial effects of corticosteroids are still unknown and different dosage patterns may be better than daily administration. Hopefully, new studies will continue to generate more answers.
Journal of Child Neurology 25(9) and colleagues studied a variety of factors that influence walking ability through functional range of motion measures, strength testing, formal gait analysis, and energy consumption studies. Preliminary data has shown that normal and corticosteroid treated Duchenne muscular dystrophy patients expend the same amount of energy to do a standard walk as they age, while energy expenditure increases significantly for untreated patients. Corticosteroids decrease muscle weakness, decrease knee contractures, decrease energy consumption, and improve cardiac and pulmonary function. This combination of effects likely affects the ability to walk. Scoliosis is another area of Duchenne muscular dystrophy that corticosteroids have affected. Scoliosis in Duchenne muscular dystrophy is because of a lack of muscular support of the growing spinal column. Once the spine is mature, as long as the curvature is less than 40 degrees, it will likely remain stable. Scoliosis affects more the 90% of untreated patients. In these patients, once curvature reaches 20 degrees, unlike in idiopathic scoliosis, it invariable progresses to over 40 degrees, which creates an unstable spine after maturity. Early spine surgery is the only answer. Patients are monitored every 6 months and once curvature reaches 20 degrees spinal surgery is offered as the curvature would predictably progress and patients are better able to tolerate intensive surgery at this earlier stage. However, corticosteroids have decreased scoliosis and changed management. In 1 study of patients treated with deflazacort, only 20% of treated patients had curvatures greater than 20 degrees compared with 80% of untreated patients. Additionally, treated patients who reached 20 degrees did not invariably progress to over 40 degrees. Therefore, instead of offering prophylactic surgery to all patients at 20 degrees, each case can be considered individually and surgery offered only after a patient reaches 40 degrees. In fact, in Dr Sussmans clinic, there has not been a single spinal stabilization operation on a patient adequately treated with corticosteroids. Lastly, Dr Sussman discussed bone health. Bone mineral density is reduced even in young patients who are still ambulatory. In untreated patients, the risk of fracture is significantly higher than in peers. One of the known side effects of corticosteroid treatment is decreased bone density, but it is unclear how this affects patients. One study found a higher fracture rate in treated Duchenne muscular dystrophy patients, while another found no difference. However, fractures are a serious issue and should be treated aggressively, as inactivity after a fracture can lead to loss of ambulation. It is also important to ensure adequate intake of calcium and vitamin D. Standing and walking stimulates bone formation and must be encouraged. Pharmacologic agents, such as bisphosphonates, may be used. The only study on these in Duchenne muscular dystrophy patients showed an increase in bone density, though whether this has an effect on fracture rate is unclear. Other agents, such as teriparatide (a recombinant form of parathyroid hormone) and denosumab, require further study. Dr Sussman closed by discussing a relatively new problem, vertebral compression fractures in steroid-treated patients. Pain is the primary complaint, and patients who experience this type
Impact of Corticosteroids on Orthopedic Management Michael D. Sussman, MD, OHSU Shriners Hospital for Children, Portland, Oregon
Dr Sussman discussed the impact of corticosteroids on orthopedic management in Duchenne muscular dystrophy. For the orthopedic challenges patients will face, including loss of walking ability, contractures, scoliosis, increased bone fragility, and growth impairment, it is important that a multidisciplinary team include a pediatric orthopedist. Loss of walking is one of the major issues seen in Duchenne muscular dystrophy. Untreated, boys with Duchenne muscular dystrophy are not able to walk independently after age 12. Studies have shown patients treated with corticosteroids walk 2.5 years longer than untreated patients. The natural wisdom attributes this to decreased loss of muscle strength. However, there may be other factors that contribute to their improved walking ability. Surprisingly, it requires very little energy and strength to stand and walk if a patient can achieve biomechanical alignment with their center of mass over their base of support. Full knee extension is able to provide knee stability without relying on quadriceps strength. The only muscle needed to walk is the gluteus maximus, because the power of walking comes from hip extension. However, when significant knee flexion contracture develops, a weak quadriceps muscle is unable to support the knee, and the patient will collapse. This may be the tipping point for losing the ability to walk. A contracture is the loss of full passive range of motion of a joint. In Duchenne muscular dystrophy patients, contractures begin extrinsically in the muscle and progress to intrinsic contractures of the joint capsule and periarticular structures. In addition to the weakness of muscles, there is infiltration of muscle with fibrofatty tissue and edema. This changes the mechanical characteristics and likely contributes to contracture development. Corticosteroids have been shown to reduce development of contracture in Duchenne muscular dystrophy patients. In 1 study, the range of motion in patients was 20 degrees greater at the knee in treated patients. Another factor that influences walking ability is energy expenditure. The generalized inefficiency of muscle function in patients, exacerbated by decreased cardiovascular and pulmonary function, results in increased energy cost. Dr Sussman
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Babcock et al of pain deserve an X-ray to look for vertebral wedging. These are not seen in untreated patients. However, it is unclear whether these vertebral compression fractures are a result of steroid treatment. Most untreated patients have had spinal stabilization surgery to prevent scoliosis and have strong metal supports along their spine that prevent vertebral compression fractures. Treated Duchenne muscular dystrophy patients may get these compression fractures because they have a straight spine that allows flexion and osteopenic vertebrae without metallic support.
1089 It is most beneficial if these are done as often as possible, so an emphasis is placed on working them into the daily routine of the family. Studies in other patient populations have shown that once a demand placed on a family exceeds 15 minutes a day, compliance decreases. To combat this, home-rehabilitation exercises are slowly built into the daily routine, and children are asked to take charge of their health and do stretches on their own. Additionally, stretches and exercises are built into other activities. Parents are encouraged to lay patients on their stomachs as they watch television to stretch hip flexion contractures and to position patients standing, rather than sitting, to play with toys. As there is only 1 randomized control trial of moderateintensity exercises, most of the recommendations concerning exercises are based on consensus opinion. The goal is to promote as much peer-related activity as possible and prevent disuse. Parents should be encouraged to let their child participate in all age-appropriate activities, if capable, with their peers. The few concerns center on eccentric activities and highintensity, high-resistance activities, which are not recommended. Additionally, patients should be monitored for signs they are overworked, such as remaining exhausted after sleep or complaining of cramps. In this case, activity should be decreased. Another goal of rehabilitation is to prolong walking and standing. This is important for bone health, for maintaining the lordotic position of the spine, and for delaying the development of scoliosis. The use of corticosteroids in these patients has decreased the need for spine surgery for scoliosis. However, when needed, bracing and surgery can prolong the ability to walk and stand by 3 to 5 years, and getting patients through their pubertal growth spurt years while standing is beneficial in maintaining bone health and preventing scoliosis. Care in the 1990s added corticosteroids and nighttime ventilation to orthopedic management and greatly changed the natural history of Duchenne muscular dystrophy. This has prolonged the ability to walk, decreased contractures and need for contracture-release surgery, decreased use of long-leg bracing, decreased incidence of scoliosis surgery, and increased survival. This has brought about a new population of adults with Duchenne muscular dystrophy. Milestones such as high school graduation, college graduation, and marriage are being attained. However, the current system is not set up to handle adult issues. A study from Denmark surveyed 65 patients, ages 18 to 42 years old, and found participants felt their quality of life was excellent and they were not worried about the future. Income, hours of personal assistance, housing, wheelchairs, and transport vans were all adequate. However, the social networks of Denmark are vastly different from what is available in the United States. Care providers must start to think about the financial costs on patients and families associated with this aging Duchenne muscular dystrophy population. While those surveyed in Denmark felt they were able to participate in desired activities, many thought they lacked an adequate education and romantic life, and 40% complained of daily pain. In 1 adult clinic in the United States, 7 of 23 patients
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Rehabilitation Care: Adapt to Change in Natural History Shree Pandya, MS, PT, University of Rochester, Rochester, New York
Ms Pandya began by describing the model of management for patients with Duchenne muscular dystrophy at the University of Rochester. The core concept of management is a multidisciplinary approach. At the University of Rochester, the core team is composed of a neurologist, nurse practitioner, and physical therapist who, along with the pediatrician, are intimately involved in the communication, education, and support of other medical providers and the community at large. The core team is supplemented by a team of therapists and ancillary staff. This level of multidisciplinary care may not be possible in more rural locations. In these instances, an interdisciplinary approach can be taken. The goal is to ensure the patients and families get the help they need. If an institution lacks a specific specialist, other personnel, under the guidance and in collaboration with a distant specialist, can provide most of the necessary care. The main goals of rehabilitation are to minimize secondary complications and maximize function and participation. Families are an integral part of the team, not only as decision-makers but also as home therapists. The first goal is to provide the family with support and education. Each family is different; care providers must respect this and be willing to meet different families at different levels. It is also important to seek family permission early to allow information exchange between the school and core team, which allows for enhanced communication and continuity of care. With the school, the core teams main role is to provide education, and may even go to the school to provide continuing education units for the teacher, gym teacher, nurse, and therapist. Physical education teachers must also be given expectations. As patients get older and can no longer participate in regular gym classes, adaptive gym-related activities, especially aquatic activities if a pool is easily accessible, should be used. The key is to avoid pulling patients out of class for services and allow them to get a proper education. In the 1980s, emphasis was placed on the physical models of rehab positioning, splinting, stretching, and other exercises designed to minimize or delay contractures. This remains one of the Care Considerations Working Group recommendations.
1090 were treated for depression. As corticosteroid use and other advancements change the natural history of Duchenne muscular dystrophy, these challenges of managing an adult population must be met. A final issue which will need to be addressed with prolonged survival is the coordination surrounding transition of care from pediatric to adult services.
Journal of Child Neurology 25(9) With evolution of symptoms of hypoventilation, nocturnal assisted ventilation, with an emphasis on noninvasive ventilation, is added. Though symptoms are of primary consideration, progression of certain indices like vital capacity falling below 30% predicted, blood or end-tidal CO2 above 45 cm-H2O or oxygen saturation below 95%, or abnormal sleep study results, are also indications. Nocturnal ventilation can then be extended into the daytime with continued symptoms. Primary indications include dyspnea, impaired swallowing, or other subtle signs of continued hypoventilation. Importantly, as a patients pulmonary status deteriorates, many will self-extend their ventilation into the daytime, and this should be asked about. With close monitoring, such as with home oximetry, these same therapies, with greater intensity or frequency, can be used to treat acute illnesses in the home setting and prevent unnecessary hospital admissions. Indications for tracheostomy were controversial and opinions varied greatly among members of the panel. However, there are good data indicating that tracheostomy-based ventilation does extend lifespan. When patients fail extubation in the intensive care unit setting or when the medical infrastructure cannot support noninvasive ventilation, tracheostomy-based ventilation is appropriate. However, there are ways to improve respiratory care infrastructure. Expertise must be developed among family members, home care providers, and inpatient staff at affiliated hospitals. This last group is vital, and difficult to initiate, as noninvasive measures are time-consuming and require increased bedside care from nursing and respiratory therapy. Dr Birnkrant then discussed some complications associated with assisted ventilation. Dr Birnkrant and colleagues looked at patients, untreated with steroids, with prolonged survival and nonpulmonary complications. Prolonged survival was defined as survival longer than 20 years. Of the 27 patients with prolonged survival, 19 had nonpulmonary complications, and of these, 18 were using assisted ventilation. Nonpulmonary complications included malnutrition due to dysphagia, which required gastrostomy, and nephrolithiasis, which required lithotripsy. These interventions have respiratory implications. In these, patients with poor baseline vital capacity, induction of anesthesia, and postoperative recovery are very high-risk and a proactive approach to respiratory management should be taken.
Guidelines of Respiratory Management David J. Birnkrant, MD, MetroHealth Medical Center/ Case Western Reserve University, Cleveland, Ohio
Dr Birnkrant discussed respiratory managements in Duchenne muscular dystrophy. Like other progressive neurological diseases of childhood, Duchenne muscular dystrophy is associated with significant respiratory complications, and death is usually because of cardiopulmonary causes. When vital capacity falls below a liter, untreated 5-year survival is 8%. However, treatment with assisted ventilation has increased survival and played a significant role in increasing mean survival from 20 years to 25 to 30 years. Other respiratory managements that played a role include mucus clearance therapies with mechanical and manually assisted coughing, lung volume recruitment, and deep lung inflation. Additionally, other therapies that played a role in increasing survival, including glucocrticoid treatment, orthopedic management, treatment of cardiomyopathy, and maintaining adequate nutrition, have also had a pulmonary impact. The recent CDC Care Considerations for Duchenne muscular dystrophy put forth recommendations, based largely on expert consensus, for respiratory management strategies stratified by disease stage. The key concept in the consensus is that proactive management leads to optimal outcomes. Most importantly, symptoms must be elicited from patients, and subtle warnings must not be ignored. For example, frequent episodes of bronchitis may reflect an underlying ineffective cough, and slipping grades or increased fatigue may reflect hypoventilation. The toolbox developed for respiratory monitoring uses little more than what might be found in an asthma clinic and includes seated forced vital capacity, maximum inspiratory and expiratory pressures, peak cough flow, oxygen saturation on room air, and end-tidal CO2. These should be measured at each visit, and as the disease progresses and symptoms arise, or vital capacity falls below 40% of predicted, gas exchange during sleep should be measured. Additionally, a home oximeter should be considered with evidence of ineffective cough, based on peak cough flow thresholds. Assessment results are then used to create a sequential approach to therapies. The first step, once vital capacity falls below 40% predicted, would be deep lung inflation and lung volume recruitment. Next, as patients demonstrate evidence of ineffective cough, manual and mechanically assisted coughing devices are added. Thresholds for this are maximum expiratory pressures below 40 cm-H20, baseline peak cough flows below 270 L/min while ill, or below 160 L/min at any time.
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Audience Member: Whats the youngest [age] you would start prednisone? I have a 15-month-old who was found more or less accidentally to have Duchenne muscular dystrophy. Should I treat him? Dr Biggar: Thats a very interesting question and there is no clear answer to it, but this is what we did about 5 years ago because we had a couple of 1-year-olds that were diagnosed because of family history. I sent an e-mail around to all of my buddies, many of them are in this room, and the consensus from them was they wouldnt start until there are some clinical
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symptoms. And that usually is around 3, 4, or 5 [years of age]. So, I think the consensus from that e-mail a few years ago was not at that age, unless theyre really severely affected and very weak. Dr Bushby: The care guidelines said that it would not be indicated under age 2 and that you should follow them for the plateau phase. So you should get a good set of functional assessments going for this child however young they are and also ask the parents about their improvement in capabilities, and when they reach the plateau is when you should start. So the consensus not only from Dr Biggars e-mail, but also from the consensus guidelines, was that there are no data yet to base starting so young.
1091 retain remarkable function. This is because the key structures for dystrophin, the binding sites for actin and beta-dystroglycan, are preserved in the micro-dystrophins. To study the function of these micro-dystrophin cassettes, adeno-associated virus/micro-dystrophin vectors were injected into dystrophin/utrophin double knockout mice. Utrophin is very similar in structure to dystrophin, and these double knockout mice offer a very severe model of disease. Dr Chamberlain and colleagues found that treated mice had a 75% reduction in creatine kinase levels and a 120% increase in specific forcegenerating capacity of the diaphragm muscle. Remarkably, the mean lifespan was 5 times greater in treated mice, increasing from about 12 to 14 weeks to 40 to 50 weeks. Of note, the treated mice began to die around a year. While most of the muscles remained normal, these animals developed esophageal hypertrophy, indicating poor penetrance of the adeno-associated viral vector into the esophagus. This is not seen in treated dystrophin knockout mice and is hopefully because of the absence of utrophin in the double knockout mice, which does not occur in Duchenne muscular dystrophy. Importantly, this is the only treatment method that has been shown to increase lifespan in an animal model of Duchenne muscular dystrophy. One final challenge with systemic delivery of viral vectors, which has been the Achilles heel of the gene therapy field, is the potential for the viral vector to elicit an immune reaction. A cellular immune response against the vector would eliminate any cells that take up the vector and would cause any treatment effect to be transient. The adeno-associated virus vectors do not contain viral genes. They are composed of only 2 components, a micro-dystrophin gene and a viral protein shell. As most Duchenne muscular dystrophy patients have null mutations, and do not make any dystrophin, it is theoretically possible that the micro-dystrophin protein would be recognized as a foreign antigen and stimulate an immune response. Utrophin is similar in structure and action to dystrophin, and studies have shown that a high-level expression of utrophin in a dystrophin knockout mouse prevents dystrophy. As it is already present in Duchenne muscular dystrophy patients, delivery of utrophin would circumvent this problem. Miniaturized versions of the utrophin protein were made in a similar manner as microdystrophin and encapsidated in adeno-associated virus vectors. A study of micro-utrophin treated double knockout mice showed that treated mice gained weight and had a lifespan equivalent to control mice. The other potential component that would elicit an immune response is the viral capsid. Studies have shown that this capsid breaks down after entering a cell, is degraded, and is cleared from the body in 3 months. Therefore, any immune response to the capsid would take place within this time. There are several contrasting studies, some of which have shown that adeno-associated viral vectors can cause a transient immune response. With a short-term course of immune suppression, it would theoretically be possible to block an immune reaction to the viral capsid, while it is cleared from the body. This was studied in a canine model of Duchenne muscular dystrophy. Dogs were pretreated with antithymocyte globulin and treated
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EMERGING NEW THERAPIES

Moderator: Jeffrey S. Chamberlain, PhD, University of Washington School of Medicine, Seattle, Washington
Preclinical Gene Therapy Advances Jeffrey S. Chamberlain, PhD, University of Washington School of Medicine, Seattle, Washington
Dr Chamberlain presented advances in gene therapy in Duchenne muscular dystrophy, primarily focusing on ongoing work in his laboratory using adeno-associated viral vectors. Like other recessively inherited diseases that result in loss of function, Duchenne muscular dystrophy is amenable to gene therapy. The most commonly envisioned approach attempts to use viral vectors to deliver expression cassettes, which contain the dystrophin protein, to muscle cells. To do this effectively, some major challenges must be overcome. The first challenge is to deliver the cassettes to muscles throughout the body, including muscles involved in respiration and cardiac muscles. Adeno-associated virus is a naturally occurring human virus that has never been connected with disease. One of the advantages of adeno-associated viral vectors is their remarkable ability to target muscle throughout the body. Once the gene cassette enters a cell, it exists as an episomal plasmid and does not integrate into the chromosome. Gene expression displays long-term persistence, and recent studies in dogs and monkeys suggest this is stable for at least 7 years. In mouse studies, adeno-associated viral vectors were able to target all the muscles throughout the body with a single injection and displayed persistent expression for the lifespan of the mouse. Another challenge is because of the size of the dystrophin gene. It is the largest gene in nature, too big for most gene delivery vectors. To compound this, the adeno-associated viral vector is rather small, and only holds 4.7 kb of DNA, which is 3 times smaller than full-length dystrophin complementary DNA. Fortunately, the dystrophin gene is divided into different domains, many of which are not critical for function. In fact, 1 patient with very mild Becker muscular dystrophy, who was still walking at age 70, was found to have a deletion of the middle 50% of the gene. This led to the development of micro-dystrophins, which are approximately one third the size of full-length dystrophin and are able to fit inside adeno-associated virus capsids and
1092 with cyclosporine and mycophenolate mofetil for 4 months after adeno-associated virus/micro-dystrophin injection. A year after immune suppression ended, muscle biopsies showed very high levels of dystrophin production. This offers encouraging evidence that the immunologic challenges can be overcome. Dr Chamberlain closed by discussing some next steps in bringing this promising therapy from the laboratory to the clinic. Recent studies in scaling up from mice to dogs have shown that whole body treatment may be possible, but multiple injections at different sites with catheters may be necessary. As intravascular delivery and immune suppression methods are optimized, phase 1 safety trials have already begun with intramuscular injections.
Journal of Child Neurology 25(9) were observed in some muscles with large intermuscle variability in effectiveness. Morpholino injection also resulted in the reduction of creatine kinase level in injected mdx mice. To apply this therapy to a greater patient population, the next aim was to skip multiple exons. This was tested in a canine muscular dystrophy model. Four different morpholinos were injected and in culture produced an in-line skipping of 5 exons. A combination of 3 of these, selected for optimal effectiveness, produced increased local dystrophin levels in a dose-dependent manner when injected intramuscularly. Next, the morpholino combination was infused systemically and produced a difference in mobility, degree of muscle contractures, and dystrophin levels on muscle biopsy in a treated dog compared with an untreated control. The heart, however, was not affected and showed no increased dystrophin positivity secondary to systemic treatment. It appears that the clinical effectiveness in dogs was by stabilizing the functional level of the animal and slowing functional deterioration. To demonstrate this effect clinically in human patients, children who would be expected to decline rapidly must be identified, treated, and evaluated for stabilization. The promising aspects of exon skipping appear to be its safety. Morpholinos are not metabolized and theoretically are not immunogenic, though splicing does form new inter-exon juctional sites that may have immunogenic potential. They are easily delivered systemically and appear to effectively stabilize clinical decline. Potential problems include the short duration of effectiveness and the lack of effect demonstrated in cardiac muscle. Dr Partridge highlighted some of the remaining questions surrounding morpholino therapy. It is not known how these compounds enter cells in vivo, and this would be important to optimize continued effectiveness. Also, the specificity of exon skipping demonstrated in the murine and canine models may be decreased in human patients. Finally, a strategy must be developed to identify optimal exon sequences in humans, but this must be done through local injection as the compounds show poor tissue culture effectiveness. Two clinical trials using these compounds by local injection have been performed. Both showed positive results and systemic trials are pending. Modified morpholinos including a decapeptide are now being investigated to improve delivery to cells and boost efficacy. The decapeptide has shown some promise in being effectively delivered to cardiac muscles. These modified morpholinos, though effective, appear to be more toxic. Overall, exon skipping therapy is now entering systemic clinical trials and may prove to be truly beneficial to children with Duchenne muscular dystrophy.
Exon Skipping Treatment Terence Partridge, PhD, Childrens National Medical Center, Washington, DC
Dr Partridge discussed exon skipping, an endogenous gene therapy. The knowledge that the functional aspects of the dystrophin gene are near the ends of the molecule, and most disease-causing mutations are in the middle section, has led to the idea that some exons may be dropped from a gene transcript that can still produce a functional product. The dystrophin exons constitute approximately 2% of the gene, and the most common mutation in Duchenne muscular dystrophy is a deletion, which, depending on the way it affects the reading frame, can lead to a nontranslatable transcript. It appears that some forms of Becker muscular dystrophy are associated with deletions that result in a translatable transcript, thus resulting in a milder phenotype. Early data from the laboratory of Dr Kay Davies showed that a patient missing exons 17 through 49 produced a relatively functional protein. From this came the idea of selectively skipping exons to shift a nontranslatable reading frame into a translatable sequence. Exon skipping is performed using antisense oligonucleotides that bind selected splice sites, leading to exon exclusion from the transcript. DNA and RNA are rapidly degraded in vivo and are not good candidates for splice site binding, so modified oligonucleotides called morpholinos have been developed. In mdx mice, which have a nonsense mutation in exon 23, the administration of 2 oligonucleotide constructs produced a transcript lacking this exon. One difficult aspect of this technology is that the constructs may bind to additional splice sites targeting not only exons containing mutations but unanticipated others. Constructs injected intramuscularly in mdx mice showed dystrophin reconstitution on Western blot analysis lasting as long as 12 weeks after treatment. Although this local effect is encouraging, a systemic delivery mechanism is necessary for true clinical value. When delivered intravenously, the antisense oligomers showed a much lower dystrophin reconstitution level. Morpholinos, more stable compounds that are not degraded in the body and are therefore apparently nontoxic, demonstrated systemic effectiveness in all muscles except cardiac. Dystrophin levels up to 50% of normal
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PTC 124: Stop Codon Treatment in Duchenne Muscular Dystrophy Richard S. Finkel, MD, The Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania
Dr Finkel discussed treatments targeted at the genetic level of disease, specifically targeting the responsible mutations rather
Babcock et al than treating a clinical syndrome. He focused on methods to bypass a premature stop codon. Many dystrophin mutations involve large deletions or duplications, but approximately 13% of mutations in Duchenne patients have a single base pair change that forms a premature stop codon. A small amount of dystrophin is produced even from sequences with these nonsense mutations, but it is not enough to prevent the Duchenne muscular dystrophy phenotype. Dr Finkel noted that although bypassing premature stop codons is currently being studied in Duchenne muscular dystrophy and cystic fibrosis, a variety of genetic disorders are mediated through such a mutation, and this therapy may have broad applications. Interest in this approach arose from early studies published in 1979 showing that aminoglycosides can suppress nonsense mutations in eukaryotic cells. The murine muscular dystrophy model, the mdx mouse, has a single base pair nonsense mutation that is responsible for its phenotype, which makes it an appropriate study model. Initially, gentamycin-treated mdx mice were found to have increased dystrophin expression as well as increased expression of gamma-sarcoglycan, a transmembrane dystrophinassociated protein. Clinical trials of gentamycin in Duchenne and Becker muscular dystrophy patients followed the murine studies and yielded differing results as to whether treatment increased dystrophin expression in muscle tissue. Interest in developing clinically effective aminoglycosides to increased dystrophin expression is ongoing, but the toxicity of these agents is a major drawback. Because of the challenges of aminoglysides, high throughput screening was applied to identify other potential compounds that were similarly effective in the mdx mouse model but orally bioavailable. One compound, PTC 124 (ataluren), was brought to clinical trial because of its effectiveness and favorable safety and toxicity profiles. This molecule induces a read-through of premature stop mutations by interacting with the mRNA bound 60S ribosomal subunit. Importantly, it does not encourage bypass of normally occurring stop codons. In the mdx mouse, ataluren improves dystrophin expression both in individual muscle fibers in vitro and in orally treated mdx mice in vivo. Effects were noted in a variety of muscles in the in vivo model, including the diaphragm and cardiac muscle. Oral treatment also demonstrated a reduction in creatine kinase level and in contraction-induced muscle injury. After establishing effective levels as well as safety and toxicity profiles in the mouse, a phase 1 clinical study was done in healthy volunteers. Ataluren was then tested in a small phase 2 study for efficacy. In vitro studies showed that 100% of patients produced some degree of dystrophin in response to treatment. The compound was given orally 3 times daily for 4 weeks to patients with a variety of premature stop mutations. Muscle biopsies and muscle enzyme studies were performed before and after treatment. Biopsies performed after 28 days of treatment showed a qualitative increase in dystrophin expression in approximately half of the subjects. This was unrelated to the type of mutation, ataluren dose, or to concurrent steroid
1093 treatment. Ataluren-associated decreases in serum creatine kinase were also observed at each dose level. Finally, clinical measures, including timed Gowers, 4-step climb, and 10meter run, were not significantly improved, but patients and their parents reported subjective symptomatic improvements. No toxicity or safety issues arose during the treatment period. In the next phase of this study, which was still blinded at the time of this meeting, the same patients were enrolled in an extended treatment protocol. This was a 48-week international randomized placebo-controlled double-blind trial with 174 patients with Duchenne or severe Becker muscular dystrophy. The primary outcome measure of this study is the distance traveled during a 6-minute walk, as this is a clinically meaningful and reproducible functional measure. Other measures include daily step counts, serum creatine kinase levels, digit span as a measure of cognitive function, and muscle biopsies for dystrophin expresssion. Though the clinical data from this trial were not yet available, no toxicity or safety concerns were noted throughout the study period. Dr Finkel concluded by summarizing that ataluren restores some degree of dystrophin expression by promoting readthrough of premature stop codons in Duchenne muscular dystrophy. Clinical trials are underway to determine the significance of this effect. Lastly, he stressed the idea that this type of intervention has broader applications for other genetic disorders caused by premature stop-codon nonsense mutations.
Other Clinical Trials and Novel Therapies in Progress in Duchenne Muscular Dystrophy Robert Leshner, MD, Childrens National Medical Center, Washington, DC
Dr Leshner discussed other potential disease-modifying therapies currently under investigation for the treatment of patients with muscular dystrophy. He began by noting that a combination of therapies, including gene therapies, may be necessary to obtain a maximum clinical result in patients affected by these disorders. Additionally, he reiterated that none of the therapies currently under investigation are likely to be curative. By understanding the mechanisms of muscle breakdown in boys with Duchenne muscular dystrophy, we can identify downstream treatment targets. These mechanisms include calcium entry into muscle cells, immunologic factor effects like those of tumor necrosis factor and transforming growth factors, the nonlocalization of nitric oxide synthetase, which may contribute to poor myocyte perfusion, and impaired muscle regeneration. The effects of dystrophin deficiency on all of these downstream mediators are not yet clearly understood. Dr Leshner proposed that we must target these downstream pathways in addition to developing therapies that increase dystrophin synthesis. Inflammatory changes observed in muscle biopsies of children with muscular dystrophy provided the original rationale for high-dose steroid treatment. These inflammatory changes are now believed to be a reaction to muscle breakdown rather than its cause, but the effectiveness of corticosteroid treatment may
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1094 in fact be in part via reduced inflammation. Abnormal calcium entry into myocytes mediates cell damage by activating intracellular proteases and lipases. The mechanism of increased calcium influx represents another potential target. Duchenne is initially marked by hypertrophic myocytes that are eventually replaced by fibrous tissue and fat. Preventing this deposition of fibrous and fatty tissue is important, as it seems that once it has occurred, the process is irreversible. Finally, free radical accumulation and mitochondrial abnormalities may also be investigated. Dr Leshner encouraged clinicians to enroll their patients in available clinical trials as much as possible but reminded us of the necessity of animal research. A new murine research facility housing 700 animals, all models for various neuromuscular diseases, is now available. This facility is developing clinically relevant outcome parameters in mice, including echocardiography, treadmill assays, front and hind paw strength measures, and activity monitoring. Standardized, clinically relevant measures for murine outcomes must be developed to optimize preclinical therapy investigation. Dr Leshner also touched on therapeutic agent selection. Though thousands of molecules have been screened as potential therapeutic agents for these diseases, the corticosteroid medications are the only lasting proven therapy. Future clinical trials must offer patients the option of combining steroids with experimental agents, as many parents and patients will not accept study designs that withhold prednisone. Quality-of-life measures, in addition to clinical outcome measures of function, objective strength, and serum proteins, must be identified and used to guide therapy development. Dr Leshner suggested that new studies investigating the natural history of Duchenne muscular dystrophy are necessary as steroids, pulmonary care, and cardiac surveillance have likely altered this significantly. A natural history study of 347 patients with Duchenne muscular dystrophy will be correlated with genotype and disease progression based on a variety of functional measures to produce a database of natural history and genetic information. Additionally, data on single nucleotide polymorphisms in potential disease-modifying genes such as osteopontin, which is a cytokine that predicts therapeutic response to exercise, will be collected. Though many promising therapeutic advances are under clinical investigation, Dr Leshner again emphasized that 1 compound or approach is not likely to be curative.
Journal of Child Neurology 25(9)

have been 1 or 2 reports in the literature over the last year claiming that adeno-associated virus vectors do get into motor neurons but, again, we have not been able to reproduce that in our laboratory. We also dont see any gene transfer to the brain. These vectors do not cross the blood-brain barrier. On the other hand, if you go intracranially with the direct injection we get very beautiful transduction of glial cells and neurons, but thats a whole different ballgame there. Audience Member: We have heard a couple of times today about the heart. We heard this morning that the natural history of heart involvement is sort of a delayed effect within the disease and then you alluded to the fact that maybe the morpholinos arent working in the heart because, maybe Im reading more into what you said, but it sounded like because the heart isnt as diseased. Did I hear that right? Dr Partridge: No, actually, we dont know why. I think its probably more profound than that. I think there is some idea that you can only get into the skeletal muscles if they are diseased, certainly there is some evidence favoring the idea that you get better entry of these oligonucleotides into diseased than nondiseased muscle. Not uncontested, I should say to that. But with the heart, we dont know why and I think that the modified morpholinos do promise to take us around on that problem if we can hit the right dose. Audience Member: In your animal model, in the canine model, is there any disease in the heart? Dr Partridge: Oh, yes, and in the mouse model. You get the same sort of infarcts, usually on the left ventricle, I think, in both species and it is often hard to work out why a mouse died because you only find it is a half-eaten mouse the next day, but dogs often survive into middle age and then die of heart failure, Im told. Is that right? Audience Member: Yes, thats my understanding. The mouse model does develop a progressive cardiomyopathy but in the dog its really quite severe. Audience Member: Dr Chamberlain showed us that newly produced dystrophin is immunogenic. I wonder, is this of concern also with the read-through dystrophin product? Dr Finkel: This is a very important question. The concern with exon skipping is youre going to make a protein that is totally naive to the patients and it is potentially immunogenic. Does this same situation potentially apply with ataluren? And my answer is no, I dont think so because these stop mutations are a little bit leaky. These patients make a small amount of fulllength protein. It is very hard to see that on the slide I showed you but with highly sensitive immunostaining, we saw that in most of the patients there is a very low level of expression. So its not a complete null mutation as you might think it would be, but Im hoping that because of this low-level expression, they are tolerized and are going to see the full-length dystrophin created by the drug as being immunogenic. Audience Member: What is your theory on how the ataluren differentiates normal from abnormal stop codons? Dr Finkel: Again an important question. The reason we think that theres this important difference and why ataluren does not affect the normal stop is because theyre read on different parts of the ribosome. The 60S subunit of the ribosome is what seems to be where the ataluren attaches and helps read-through these

Audience Member: Have you looked at the other tissues, such as motor neurons or Schwann cells? Dr Chamberlain: Yes, we have. Using adeno-associated virus 6, which is the primary serotype that we use, we have not been able to detect significant delivery to motor neurons or Schwann cells. We have been a little disappointed in that because we would like to branch this out toward ALS [amyotrophic lateral sclerosis] and other disorders. We have spent a lot of time looking at that and were not convinced that were getting any significant transfer to motor neurons. Now, there 1094
Babcock et al
premature stop mutations, whereas a totally different portion of the ribosome is responsible for reading the normal stop and the poly A tails at the end of the message. Audience Member: Im wondering whether the PTC124 can penetrate the blood-brain barrier. Dr Finkel: It does to a small degree in the mouse model, not well, and the expression in heart is not as good as skeletal muscle, but it does to some degree. The question is what does that do in humans, and we dont know that. Dr Leshner: Dr Mendell, would you mind giving an update on your gene therapy protocols? Dr Mendell: We have done 2 gene therapy trials. In the Duchenne trial, weve had immunogenic problems with the dystrophin gene. If we express the trans gene in an area of deletion, we see a T-cell immune response. The one new finding that I think is very novel is that we found that revertant fibers express new epitopes, and the new epitopes are immunogenic. The second trial we did was in limb-girdle dystrophy in which we have mostly missense mutations and a baseline gene expression with spontaneous readthrough, and protection against this transcript, with stable transcripts that escape nonsense-mediated decay. In the gene therapy trial, we have had very good results with missense mutations and in gene transfer because there was no immunogenic response to the newly expressed protein. Dr Leshner: Thank you so much. Dr Muntoni, would you give us a little update on the exon 51? Dr Muntoni: As you know, last year we performed an intramuscular study where antisense oligonucleotides were given to 1 of the foot muscles of the children, the EDB [extensor digitorum brevis] muscle, and the contralateral muscle was given a saline control. The result of that study had been published in Lancet Neurology. After this early study, we started in February 2009 a dose escalation study where systemic morpholino antisense oligonucleotides are administered to a group of children with Duchenne muscular dystrophy. The group is divided into 6 different categories of dose escalation and each child receives 12 weeks intravenous administration of the antisense oligonucleotides. This is a study that is funded by the Medical Research Council with contribution from AVI as well. The study is now recruiting cohort 5 of the 6, and so far the drug has been well-tolerated. We hope to have completed the last trial in cohort 6 by late February or very early March 2010. Dr Leshner: And the cohort 6 will be the 20 mg/kg of morpholino IV [intravenously]? Dr Muntoni: Thats right. At the moment we have completed the third cohort that is the 2 mg/kg. We have nearly completed the fourth cohort, the 4 mg/kg. We are in the middle of the fifth cohort that is the 10 mg/kg and we will start recruiting the first patients in cohort 6 at the 20 mg/kg dose in late November. Audience Member: While Ive enjoyed this talk extremely, what I understand is that were trying to move our Duchenne muscular dystrophy patients toward milder phenotypes like the Becker muscular dystrophy type. Do we know the genotypephenotype correlations of what makes a Becker versus a Duchenne muscular dystrophy at the gene level? Dr Chamberlain: I think there have been a lot of genotypephenotype correlations because really since the discovery of the dystrophin gene, there have been a tremendous number of
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primarily deletion-based studies where you can go in and identify what mutations are associated with Becker as opposed to Duchenne, and there is a whole spectrum of severities associated with Becker muscular dystrophy. I think the primary correlations that have come out have been from the interframe deletions, where you can make correlations between which portions of the gene are missing and whether you get a milder or more severe Becker phenotype. There have been fewer studies, although those are equally important, identifying that some mutations can affect the stability of the dystrophin protein such that even though you have a fairly large dystrophin protein, its not being produced at the levels you might see in a wild-type individual. This can also have an impact on the severity of the Becker muscular dystrophy. This is not to say that we dont need more of that information because I think thats ultimately going to be important in optimizing a lot of the different types of therapy that we talked about today. For example, when I was talking about trying to generate micro-dystrophins with increased functional capacity, we rely heavily on the clinical literature to see what mutations are already associated with milder phenotypes and then we try to generate variations on that, creating new synthetic dystrophins that have never been seen in nature and testing those out in the mdx mouse. The exon skipping strategies are also dependent on knowing is there a natural history of seeing patients that have those exons skipped due to deletion, and what type of phenotype do they have compared to the full-length dystrophin? I think your point is well taken that the structure function studies and how they relate to different mutations are going to be very important in guiding the development of these therapeutics and gauging their outcome. Audience Member: Yes, when you get a dystrophin gene result, you want to be able to tell the patients the prognosis and not all in-frame mutations give you a milder phenotype. And my second question is, are there studies that follow other biomarkers like creatine kinases? I know that they are elevated at certain times; is that something that can correlate with disease severity? Can we use it along with Hammersmith or other functional tests to determine whether theyre at a plateau phase? Dr Chamberlain: Creatine kinases should be fairly useful. Dr Leshner: Yes, the creatine kinase is 1 parameter of muscle breakdown and one would expect that if you significantly diminish myonecrosis, that you would be rewarded with a lowering of the creatine kinase or generating muscle cells which hopefully do not have the leaky membranes and are not undergoing active destruction. I do not know how good this is as a marker for the ultimate functionality of the individual, and it would certainly never be used as a primary endpoint in any clinical trial. Dr Moxley: One additional comment. There is always the challenge of knowing all the components which influence the clearance as well as the release of creatine kinase. If you were to look at Dr Finkels paper that I cited, CK [creatine kinase] goes up as the patients increase physical activity. Its hugely up in marathon runners. Im just suggesting that its a work in progress to know how to use creatine kinase. It may be simpler to choose another surrogate marker, which Im not sure we have at this point. Audience Member: I am wondering, given the long track record of molecules that seem to have some improvement in 1095
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mdx but have not panned out clinically, how you foresee prioritization molecules given the limited number of patients and the potential of several molecules being successful in mdx mice but not necessarily being successful in patients? Dr Leshner: First, I think that the far more detailed functional testing which is going on in mice now will probably give us a better idea than some of the older means of testing; the running down a ramp with a cord tied to the back of a rats tail and seeing the force generated is not quite as physiologic as watching the mice on the treadmills, measuring their activity over prolonged periods of time. The other thing is that the mouse remains a good starting point for looking for your preclinical toxicology studies prior to human trials. With some therapeutic strategies, this may be exquisitely species-specific, and although compounds may be safe in mice, the important question is whether or not there are toxicities in young men with Duchenne dystrophy. So the mouse remains valuable, but all of us realize the limitations of murine testing. Dr Leshner: The question is whether or not every compound should be put through a larger animal test such as a canine model, and I guess everyone has their own prejudice. I feel that its still logical to start in the murine model and for many of the therapies I think that a second-level testing, for instance the dog, would certainly be a very logical transition to human testing. But again, starting off in dogs would be cumbersome and extraordinarily expensive. Dr Chamberlain: Yes, if I could just add a brief comment on that. I think youre going to have to take it on a case-by-case basis depending on the treatment strategy. With things that are much more experimental and novel and have not really been applied to much of an extent in humans, its a good idea to go through the dog. The problem with dog models is there are so few of them, theyre very hard to breed, theyre very expensive, and if we had a requirement that everything had to go through dog testing before it could come to the trials, it would just tremendously slow down the pipeline. So I think we need to be a little careful about that.
Journal of Child Neurology 25(9) dystrophy is one of the first identified in this group of disorders, but the genetic cause of this disease is yet unknown. In Duchenne dystrophy, international research efforts such as TREAT-NMD are necessary to coordinate and strengthen clinical trials. The MD STARnet program, discussed by Dr Mathews, aims to distribute resources for muscular dystrophy patients throughout previously underserved communities. This idea of delivery of interventions was also discussed by Dr Birnkrant, who highlighted the difficulty of implementing evidence-based pulmonary interventions in muscular dystrophy clinics. This could be improved with increased collaboration between neurologists and pulmonologists. Abundant data presented about the use of steroids showed clear positive effects on walking ability, respiration, contractures, and survival of muscular dystrophy patients. Further work is needed to compare different steroid regimens, and a new study focused on this is underway. Although steroid use has greatly decreased the need for corrective scoliosis surgery, knee contractures remain an important ambulation-limiting aspect of Duchenne dystrophy that seems to be related to quadriceps strength. One idea for increasing quadriceps strength is by vascular delivery of the dystrophin gene directly to the muscle. Another approach, which is just beginning to be explored in clinical trials, is the use of adeno-associated viral vectors to mediate gene repair. In the realm of new therapies, the dystrophin genes size is a major limiting factor in the development of effective gene therapy delivery systems. Another difficulty is the immunogenicity of the dystrophin gene. Utrophin, an endogenous nonimmunogenic protein and a dystrophin homologue, may provide a means of avoiding this problem. Dr Mendell showed data where a patient was immunogenic to a new dystrophin epitope prior to gene therapy, suggesting that patients may need to be screened for pre-existing immunity when gene therapy is considered. Another approach, exon skipping, prevents premature protein termination and forms a shortened protein that has a phenotypic benefit in canine models. Ataluren (PTC124) is a compound that allows polypeptide synthesis to bypass a premature dystrophin stop codon and continue. This compound is currently in a phase 2B clinical trial with 174 patients; it appears to be safe and to increase dystrophin production. Lastly, Dr Mendell reiterated that it is unlikely that any of these new treatments will work equally well in all patients, though they all hold promise.
FUTURE DIRECTIONS
Moderators: John Porter, PhD, National Institute of Neurological Disorders and Stroke Muscle Disease Program Director, and Valerie Cwik, MD, Muscular Dystrophy Association
Executive Summary for the Day Jerry R. Mendell, MD, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio
Dr Mendell gave a closing talk highlighting major points of the day and posing some remaining questions for the future directions panel. He began by discussing clinical challenges beyond Duchenne dystrophy. New treatment strategies for congenital dystrophies, including cyclosporine-like agents that can correct mitochondrial dysfunction associated with apoptosis in the collagen VI disorders, are very promising and perhaps offer benefits beyond muscular dystrophy. Fascioscapulohumeral muscular
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PANEL DISCUSSION
Moderator: Valerie Cwik, MD, Muscular Dystrophy Association; Panel: Paula Clemens, MD, University of Pittsburgh, Pittsburgh, Pennsylvania; Anne Connolly, MD, Washington University School of Medicine, St Louis, Missouri; Patricia Furlong, RN, Parent Project Muscular Dystrophy; Susan Iannaccone, MD, University of Texas Southwestern Medical Center, Dallas, Texas; and Katherine Wagner, MD, PhD, The Johns Hopkins Hospital, Baltimore, Maryland
Babcock et al The panel discussion focused on new challenges that have arisen from the changing natural history of muscular dystrophy and in the testing and application of promising new treatments. A common theme throughout the discussion was the need for careful involvement of patients and families in research to maximize participation without fostering premature expectations.
1097 It was suggested that even though approximately 25% of patients do not have clinically apparent improvements in walking ability with steroid treatment, steroids should be continued in these patients because of their potential benefits in other systems, including cardiac, pulmonary, spine, and upper extremity function.
Clinical Issues
The average age of diagnosis of Duchenne dystrophy has not changed in 20 years. The panel agreed that early diagnosis must improve. This can be done by increasing the knowledge of primary care physicians, general pediatric residents, pediatricians, and community members who interact with young children, as they are often the first to see children with motor delay. A useful approach to screening recommended by several members of the panel was watching children walk and run at each well-child visit. Neck flexor weakness is also a helpful early sign of weakness in affected children that is easily evaluated in the clinic. Lastly, it is recommended that any suspicion of a myopathy should warrant checking a creatine kinase level. Development of a neonatal diagnostic test to be administered on a state-by-state basis was also suggested as a strategy to improve early diagnosis. This will become more relevant as additional therapies are available for early intervention in muscular dystrophy. The panel emphasized that quality of life must be a major consideration in the development of new therapies and the use of currently available treatments, like steroids. A balance is necessary between the stabilizing effects of steroid use and their significant side effects. Also, as patients are now entering adulthood, clinicians must become advocates to encourage their full participation in society. New, less-intensive study designs and more appropriate outcome measures will also be necessary as interventions are studied in an older population because transportation and evaluation become more cumbersome for patients, and measures such as the 6-minute walk are no longer applicable to nonambulatory patients. As patients approach adolescence, their complex needs necessitate interdisciplinary treatment. This can be optimized by organizing interdisciplinary clinics during which patients are seen by a variety of specialists, including neurology, pulmonology, cardiology, physical therapy, and others, all in 1 visit.
Emerging Therapies
The panel highlighted the idea of parents as partners in research and as the driving force for translation of basic findings to applicable therapies. It is important to communicate accurately with families and to refrain from suggesting that any therapy currently being investigated will be curative or even effective for all children with Duchenne dystrophy. With several prospective therapies to increase dystrophin production, it will be important to know what level of dystrophin production is needed to produce a meaningful clinical effect. Also, what is the best way to measure dystrophin and how frequently should this be done in affected children who are study participants? Lastly, with systemic administration of therapy, how will production vary between muscles and how can we avoid variability in sampling? More work must be done to understand fully the phenotypic consequences of truncated dystrophins, as in-frame mutations lead to varying severities of disease in Becker muscular dystrophy, so not all of these are ideal therapeutically. Overall, the panels message was a positive one. Lifeexpectancy of patients with Duchenne muscular dystrophy has increased substantially in recent years with the development of less invasive and more effective interventions. Future strategies, including gene therapy, hold a great deal of promise for improved quality of life for these boys and young men and may eventually lead to a cure.
Acknowledgments
Presented at the Neurobiology of Disease in Children Symposium: Muscular Dystrophy, in conjunction with the 38th Annual Meeting of the Child Neurology Society, Louisville, Kentucky, October 14, 2009. Special thanks to Dr John D. Porter from the National Institute of Neurological Disorders and Stroke for assistance in planning the conference. The authors would also like to thank Melanie Fridl Ross, MSJ, ELS, for editing this manuscript.
Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the authorship and/or publication of this article.
Corticosteroid Treatment
A better understanding is necessary of the ideal time to start corticosteroid therapy. Improved understanding of the natural history of disease in very young corticosteroid-treated patients may guide a definition of the lower age limits of treatment initiation.
Funding
The authors disclosed receipt of the following financial support for the research and/or authorship of this article: Supported by grants from the NIH (5R13NS040925-09), the NIH Office of Rare Diseases Research, the Muscular Dystrophy Association, and the Child Neurology Society.

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Muscular Dystrophy: New Opportunities for Diagnosis and Treatment

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Muscular Dystrophy: New Opportunities for Diagnosis and Treatment

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EMERGING NEW THERAPIES

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Journal of Child Neurology 25(9)

Questions and Answers

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Declaration of Conflicting Interests

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