Dystrophinopathies Background Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy affecting 1 in 3500 boys born

worldwide. Although the name Duchenne is inextricably linked to the most common childhood muscular dystrophy, it was Gowers who recognized Sir Charles Bell for providing the first clinical description of Duchenne dystrophy in his 1830 publication, The Nervous System of the Human Body. Others, including Edward Meryon in 1852 and John Little in 1853, described families of boys with delayed motor milestones, calf enlargement, progressive inability to ambulate, heel cord contractures, and death at an early age. In an 1868 publication, Duchenne established the diagnostic criteria that are still used. These criteria include (1) weakness with onset in the legs; (2) hyperlordosis with wide-based gait; (3) hypertrophy of weak muscles; (4) progressive course over time; (5) reduced muscle contractility on electrical stimulation in advanced stages of the disease; and (6) absence of bladder or bowel dysfunction, sensory disturbance, or febrile illness. Gowers was the first to deduce the genetic basis for the disease and the first to describe patients with delayed onset of disease. In 1962, Becker proposed that the less symptomatic patients reflected milder mutations in the same gene. These patients are now classified as having Becker muscular dystrophy (BMD). In 1986, exactly 100 years after Gowers' keen observations, Kunkel identified the Duchenne muscular dystrophy gene located at band Xp21 and provided molecular genetic confirmation of the X-linked inheritance pattern. The Duchenne muscular dystrophy gene was named dystrophin. It is the largest recorded human gene encoding a 427-kd protein, dystrophin. Dystrophin plays an integral role in sarcolemmal stability. Research by Ervasti as well as Yoshida and Ozawa in the 1990s shed further light on the complex association of the dystrophin protein with a number of transmembrane proteins [1, 2] and glycoproteins, referred to as sarcoglycans and dystroglycans. Another similar 395-kd protein, known as utrophin, has also been identified. This protein has a similar structure to dystrophin and seems able to perform some of the same functions. Despite there being no cure for the dystrophinopathies, knowing the genetic cause and related functions of dystrophin has been invaluable in creating new molecular and pharmacologic techniques for diagnosis and treatment. Pathophysiology Dystrophin protein is integral to the structural stability of the myofiber. Without dystrophin, muscles are susceptible to mechanical injury and undergo repeated cycles of necrosis and regeneration. Ultimately, regenerative capabilities are exhausted or inactivated. In the 1850s, Edward Meryon used a small harpoonlike device to perform muscle biopsies and described the tissue from an affected patient: "The striped elementary primitive fibers were completely destroyed. The sarcous element being diffused, and in many places, converted into oil globules and granular matter, whilst the sarcolemma or tunic of the elementary fibre was broken down and destroyed." In order to understand how a mutation in the gene can cause such devastation, accurate conceptualization of the structure of dystrophin is necessary. Dystrophin protein is encoded by the largest gene described to date. It occupies almost 2% of the X chromosome and nearly 0.05% of the entire genome. The gene consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. It is expressed mainly in smooth, cardiac, and skeletal muscle, with lower levels in the brain. In muscle, dystrophin is expressed as a 427-kd protein that consists of 2 apposed globular heads with a flexible rod-shaped center that links the intracellular actin cytoskeleton to the extracellular matrix via the dystroglycan complex. The protein is organized into 4

structural domains including the amino-terminal actin-binding domain, a central rod domain, a cysteine-rich domain, and a carboxy-terminal domain. Its amino terminal end insinuates with the subsarcolemmal actin filaments of myofibrils, while cysteine-rich domains of the carboxy-terminal end associate with betadystroglycan as well as elements of the sarcoglycan complex, all of which are contained within the sarcolemmal membrane. Betadystroglycan in turn anchors the entire complex to the basal lamina via laminin.

Structure of the dystroglycan complex (adapted from Ozawa et al).

The molecular organization of integral and peripheral components of the dystrophin-glycoprotein complex and novel proteins involved in muscular dystrophy in skeletal muscle. Deletions or duplications of the dystrophin gene that do not disturb the reading frame may lead to minor alterations in the protein structure, and by extension, the function of dystrophin particularly, particularly if in-frame changes are located within the aminoterminal or central regions. In contrast, mutations that disturb the reading frame, including premature stop codons, produce a severely truncated, completely dysfunctional protein product or no protein at all.

Point vs frameshift mutations. In contrast to most point mutations, which generally preserve the reading frame, frameshift mutations often lead to truncated protein products. The functional loss of dystrophin protein initiates a cascade of events, including loss of other components of the dystrophinassociated glycoprotein complex, sarcolemmal breakdown with attendant calcium ion influx, phospholipase activation, oxidative cellular injury, and, ultimately, myonecrosis. Microscopic evaluation in the early stages of the disease reveals widespread myonecrosis with fiber splitting (see image below). Interspersed between the dying myocytes are ghost cells, the shells

of formerly healthy tissue. Inflammatory cell infiltration of the necrotic fibers may be observed in particularly aggressive areas of muscle biopsies. Fibers that survive exhibit considerable variability and often demonstrate internal nuclei. As the disease progresses, dead muscle fibers are cleared away by macrophages and replaced by fatty and connective tissue elements, conveying a deceptively healthy appearance to the muscle (pseudohypertrophy), especially calves and forearms.

Rarely, skewed random inactivation of healthy copies of the X chromosome leads to the Becker/Duchenne phenotype in females who carry the dystrophin mutation. Females with Turner syndrome (XO) or uniparental disomy or those who have translocations between the X and autosomal chromosomes may similarly manifest the Duchenne phenotype. Elevations of creatine phosphokinase (CPK) level are found in two thirds of female carriers, the vast majority of whom are clinically asymptomatic. Duchenne muscular dystrophy clinically manifests in patients aged 3-7 years, with development of lordosis, a waddling gait, and the Gowers sign. Calf pseudohypertrophy follows 1-2 years later. Most patients are wheelchair bound by age 12 years. Becker muscular dystrophy follows a much more variable course, manifesting any time from age 3 years to late adulthood. Waddling gait, manifesting in children aged 2-6 years, is often the first symptom in patients with Duchenne muscular dystrophy and is secondary to hip girdle muscle weakness. Sometimes a young boy may come to medical attention because of elevated liver function enzymes (AST, ALT), and in such cases serum creatine kinases CK and GGT levels should be checked prior to considering liver biopsies. Occasionally a young boy may be referred for speech delay or learning issues, but he may turn out to harbor a dystrophin mutation. Most children with dystrophinopathy have IQs about one standard deviation lower than the general population, but certainly plenty of exceptions exist. The low intellectual skills, such as cognitive issues (learning differences, attention deficit hyperactivity disorder, obsessive-compulsive disorder, pervasive developmental disorder, mentalretardation), are seen in up to 30% of patients with dystrophinopathy. Children with Duchenne or Becker muscular dystrophy perform particularly poorly on tests of verbal skills and have [3] challenges in processing complex verbal information. In some older boys or young men, dilated cardiomyopathy findings may lead to provincial diagnoses such as viral or idiopathic cardiomyopathy when in fact a dystrophin mutation may be the underlying reason. Because of proximal lower back and extremity weakness, parents often note that the boy pushes on his knees in order to stand; this is known as Gowers sign.

Age

Dystrophic muscle (A = Gomori trichrome; B = hematoxylin and eosin [H&E] stain). Epidemiology Frequency United States Duchenne muscular dystrophy is by far the most common childhood-onset muscular dystrophy, afflicting 1 in 3300 boys with an overall prevalence of 63 cases per million. The prevalence of the Becker phenotype is 24 cases per million. One third of these cases are due to spontaneous mutations, while the rest are inherited in an X-linked dominant manner. Gonadal mosaicism accounts for approximately 20% of new Duchenne muscular dystrophy cases. Mortality/Morbidity Duchenne muscular dystrophy is much more than a disease of skeletal muscles. Dystrophin is also found in the heart, brain, and smooth muscle. Late-stage cardiac fibrosis can lead to output failure and pulmonary congestion, a common cause of death. Additionally, cardiac fibrosis can include cardiomyopathy and conduction abnormalities, which can induce fatal arrhythmias. Weakness of skeletal muscle can contribute to cardiopulmonary complications. Scoliotic deformity from paraspinal muscle asymmetric atrophy impairs pulmonary and gastrointestinal function, predisposing individuals to pneumonia, respiratory failure, and poor nutrition. Smooth muscle dysfunction as a result of abnormal or absent dystrophin, plus inactivity, leads to gastrointestinal dysmotility, causing constipation and diarrhea. In general, patients with Becker muscular dystrophy have much greater phenotypic variability; patients may become wheelchair bound as early as age 20 years or as late as age 70 years. Motor dysfunction usually is at least a decade later than in Duchenne muscular dystrophy. Once wheelchair bound, patients with dystrophy become much more susceptible to the scourges of the sedentary, which include scoliosis, contractures, decubitus ulcers, and impaired pulmonary function. Cardiomyopathy also occurs in patients with Becker muscular dystrophy, and conduction abnormalities may dominate the clinical picture, necessitating medications, implantation of a defibrillator, or even evaluation for heart transplant. Although significant advances have been made in understanding the molecular underpinnings of the disorder, Duchenne muscular dystrophy remains an incurable illness with a mortality rate of 100%. Like its clinical presentation, the prognosis of patients with Becker muscular dystrophy is variable, with patients who are less affected ultimately dying of other diseases after a near-normal life span. Sex

History

Duchenne and Becker muscular dystrophy almost exclusively affect males because of the X-linked inheritance pattern.

Gowers sign. The calf enlargement imparts the illusory appearance of strength, but, in fact, the enlarged calf muscles are caused

by fatty and fibrotic infiltration of degenerated muscles. This is seen in conjunction with more prominent toewalking. Sometimes an apparent pseudohypertrophy is also seen in the forearms and tongue. However, another explanation may relate to compensatory hypertrophy of the calves secondary to weak tibialis anterior muscles, which tend to be affected earlier and more prominently. Contractures Inexorable progressive weakness is seen in the proximal musculature, initially in the lower extremities, but later involving the neck flexors, shoulders, and arms. Cardiac surveillance should be implemented at time of diagnosis and should incorporate echocardiography plus ECG and pediatric cardiology expertise. Around the age of 8 years, most patients notice difficulty with ascending stairs and respiratory muscle strength begins a slow but steady decline. Approximately the time that independent ambulation is most challenged, the forced vital capacity begins to gradually wane, leading to symptoms of nocturnal hypoxemia such as lethargy and early morning headaches. Scoliosis may progress especially when more wheelchair dependent. If wheelchair bound and profoundly weak, patients develop terminal respiratory or cardiac failure, usually by the early 20s, if not sooner. Poor nutritional intake can also be a serious complication in individuals with severe end-stage Duchenne muscular dystrophy. Some families and individuals become socially withdrawn and may impact further on overall psychosocial health. Family, financial, school, community, and sibling issues can be significant. Generally, neck flexors, wrist extensors, quadriceps, tibialis anterior, biceps, and triceps muscles are affected more than the neck extensors, wrist flexors, deltoids, hamstrings, gastrocnemii, and solei. Deep tendon reflexes, which tend to parallel muscle fiber loss, slowly diminish and ultimately disappear. By age 10 years, 70% of children are hobbled by contractures of the iliotibial bands, hip flexors, and heel cords. Most are wheelchair bound by this time, creating a vicious cycle of immobility and further formation of contractures. Asymmetric weakening of the paraspinal muscles leads to kyphoscoliosis, which in turn further compromises pulmonary and gastrointestinal function. Inability to generate a forceful cough underlies the development of atelectasis with attendant episodes of pneumonia. Compared with Duchenne muscular dystrophy, the Becker phenotype manifests slower (ie, in those aged 10-20 y) and evolves over a longer period of time. Muscle weakness is milder than in Duchenne muscular dystrophy, and calf pseudohypertrophy and contractures are not invariant features. In contrast to patients with Duchenne muscular dystrophy who are wheelchair bound by age 10 years, some patients with Becker muscular dystrophy are able to ambulate independently past the fourth decade of life; some are able to ambulate into the seventh decade of life. While average life expectancy of patients with mild Becker muscular dystrophy (ie, ~40s) is diminished compared to

that of the general population, survival of these individuals into the seventh or eighth decade of their lives is not unusual. Causes Duchenne and Becker muscular dystrophy are caused by mutations in the same gene encoding dystrophin. Mutations that result in the absence or severe reduction of the dystrophin protein generally result in Duchenne muscular dystrophy, while those that lead to a less severe reduction and/or expression of an internally truncated, semifunctional protein generally result in Becker muscular dystrophy. The size of the mutation is not always a determining factor of severity. For example, premature stop codons may be a single DNA base change. There are correlations with the type of mutation, location, and severity. Deletions, duplications, and frame-shift mutations resulting in the absence or truncation of the protein are associated with the most severe phenotypes seen in Duchenne muscular dystrophy, while in-frame mutations generally lead to a less severe phenotype seen in Becker muscular dystrophy. Exceptions or clinical outliers defy these generalizations and researchers believe modifier genes may contribute. Analysis of the location of deletions has shown that the amino-terminal, cysteine-rich, and carboxy-terminal domains are essential for dystrophin function, while the central rod domain can accommodate large in-frame deletions. Larger deletions of one or more exons cause approximately 59% of Duchenne muscular dystrophy and 65% of Becker muscular dystrophy cases. Premature stop codon mutations are found in 15%, duplications in 5%, and the rest are caused by frameshift, insertions/deletions, splice site, or missense mutations. Despite the fact that most of the cases of Duchenne and Becker muscular dystrophy are transmitted in a known Xlinked manner (mother may be a known carrier), one third are the result of a spontaneous mutation with no family history. Differentials Congenital Muscular Dystrophy Congenital Myopathies Dermatomyositis/Polymyositis Disorders of Carbohydrate Metabolism Emery-Dreifuss Muscular Dystrophy Endocrine Myopathies Facioscapulohumeral Dystrophy HIV-1 Associated Myopathies Kennedy Disease Lambert-Eaton Myasthenic Syndrome Limb-Girdle Muscular Dystrophy Metabolic Myopathies Spinal Muscular Atrophy Laboratory Studies Serum creatine phosphokinase (CPK) This level is always increased in patients with Duchenne muscular dystrophy or Becker muscular dystrophy, probably from birth. It often is increased to levels that are 50-100 times the reference range (ie, as high as 20,000 mU/mL). In late stage DMD very little muscle mass remains to give rise to an elevated serum CPK level. Recognizing that the natural history of serum CPK in DMD

Physical

is known to decrease over time, especially for longer-term clinical trials, is important. A child or young adult with a CPK level within the reference range does not likely have a dystrophinopathy. Strongly suspect Duchenne muscular dystrophy in a child with proximal weakness and very elevated levels of CPK. Perform further specific diagnostic testing, including DNA mutation analysis, to confirm the underlying diagnosis (see Other Tests). Imaging Studies Scoliosis frequently ensues in patients with Duchenne muscular dystrophy, particularly after they are wheelchair dependent. Radiographs of the spine are important for screening and evaluating the degree of scoliotic deformity. As the disease progresses and dyspnea becomes a complaint, chest radiography is also likely to become a part of the evaluation. Beyond imaging for scoliosis and dyspnea, imaging studies are of little help in making the diagnosis. Imaging studies of the brain are usually unremarkable. Dual energy x-ray absorptiometry is a radiographic technique to estimate bone mineral density. Individuals with dystrophinopathies can have accelerated osteopenia/osteoporosis/fracture risk, especially longbones and vertebral compressions, due to the sedentary condition, fall risk, vitamin D deficiency, calcium intake deficiency, poor sunlight exposure, and chronic corticosteroid treatment. Other Tests

Electromyography o Electromyography (EMG), even though not diagnostic, narrows the differential diagnosis by effectively excluding primarily neurogenic processes such as spinal muscular atrophy. o In general, the proximal muscles of the lower extremities may exhibit the more prominent EMG findings. A sufficient number of muscles need to be sampled to establish the presence of a diffuse process such as a dystrophy. The more revealing findings will be obtained in muscles of intermediate involvement with respect to weakness. o The motor unit action potentials (MUAPs) in patients with Duchenne or Becker muscular dystrophy are typically of short duration, particularly the simple (ie, nonpolyphasic) MUAPs. MUAP amplitudes are variable (normal to reduced) and they are typically polyphasic from the variability in muscle fiber diameters, resulting in longer MUAP durations. Early recruitment of MUAPs may be seen. If muscle fiber loss is severe, then what appears to be a loss of motor units may be seen with fast firing individual spikes. The latter are distinguished from neurogenic processes by their generally lower-than-normal amplitudes and reduced area of spikes. o Fibrillation potentials and positive sharp waves, which represent spontaneously depolarizing muscle fibers bereft of nervous innervation, are encountered in active disease as necrosis engulfs the motor endplate or separates the endplate from other portions of the muscle fiber. These may be difficult to see in some muscles,

requiring higher-than-usual sensitivity settings on the amplifier. Molecular diagnosis o Individuals with Duchenne or Becker muscular dystrophy can be reliably and accurately detected from peripheral blood samples in nearly all cases. If uninformative deletion/duplication genetic tests have resulted, direct sequencing of the dystrophin gene is a viable option. Other innovative methods have been devised for accurate noninvasive diagnosis. Currently, most laboratories use multiplex PCR amplification to examine deletion "hotspots," which account for approximately 59% of all mutations. This method has a 98% detection rate for deletions. Duplications, which account for 5% of mutations, can be detected by several different quantitative techniques, including Southern blot, quantitative PCR, multiplex amplifiable probe hybridization (MAPH), and multiplex ligation-dependent probe (MLPA). These techniques are also highly sensitive for detecting deletions. The remaining one third of the mutations are composed of subexonic sequences, of which 34% are nonsense mutations, 33% are frameshifts, 29% are splice site mutations, and 4% are missense mutations. These mutations can be screened for by using techniques such as denaturing highperformance liquid chromatography (dHPLC); single- stranded conformational polymorphism analysis with single condition amplification internal primers (SCAIP) or detection of virtually all mutations (DOVAM), a robotically enhanced multiplexed method; or denaturing gradient gel electrophoresis. o Recently, 96% of mutations in patients with Duchenne muscular dystrophy have been shown to be noninvasively identified by using these techniques in a 3-tiered approach. Tier 1 is PCR amplification to detect large deletions, tier 2 would use DOVAM to rapidly scan for point mutations, and tier 3 would use MAPH to define duplications. Other similar techniques can be substituted for any of the tiers. For example, MAPH can be substituted with Southern blot. This same approach can also be applied to the patient with Becker muscular dystrophy. While most of these techniques were originally used for research purposes, many are now available clinically. o In patients without detectable mutations of the dystrophin gene, diagnosis requires muscle biopsy for dystrophin protein quantification (see muscle biopsy in Procedures). For some families of a young boy found to have a dystrophin mutation, the muscle biopsy can provide critically important dystrophin protein

information such as molecular weight size and abundance with a western blot. Immunolabeling of frozen muscle sections can enable epitope identification. This information can offer prognostic value if a predicted DNA mutation is in- or out-of-frame as some software modelling predictions and DNA sequencing techniques do indeed have a small error rate. Electrocardiogram and echocardiogram o Electrocardiogram (ECG) provides a simple means for uncovering sinus arrhythmias and also may demonstrate deep Q waves and elevated right precordial R waves. o Transthoracic echocardiography yields a clearer and more dynamic view of the heart, often revealing small ventricles with prolonged diastolic relaxation. o A Holter monitor is valuable for paroxysmal arrhythmias. Cardiac MRI and gadolinium enhancement are new noninvasive technologies that can better characterize cardiac tissue changes in dystrophinopathy and may implicate earlier treatments or prophylactic regimens to stabilize the heart. Carrier detection o Carrier detection is an important aspect of the care and evaluation of patients with Duchenne muscular dystrophy and Becker muscular dystrophy and their family members. o A small minority of female carriers are symptomatic, but even in these symptomatic patients, correct diagnosis requires appropriate testing. o For many years, CPK testing was the best method for carrier detection; however, it is elevated in only two thirds of female carriers and the results can be difficult to interpret in ethnic and racial groups with normally elevated CPK levels. For example, African Americans have a higher reference range than whites; CPK levels of African Americans may exceed the laboratorystated normal limits without the presence of any pathology. o In families in which an affected male has a known deletion or duplication of the dystrophin gene, testing for carrier status is performed accurately by testing possible carriers for the same deletion or duplication, the absence of which generally excludes them as a carrier. These methods can also be used in prenatal diagnosis but gonadal mosaicism does occur in less than 8% of women and a negative blood DNA tests can be falsely reassuring o If the affected males in the family are unavailable for deletion or duplication testing, the female still can be tested, but the absence of a DNA abnormality does not exclude them as carriers. Obviously, the presence of a deletion or duplication in a female always conveys carrier status. o In families in which the affected male has no detectable deletion or duplication, muscle immunofluorescence for dystrophin can be used in some cases. Carrier females should exhibit a mosaic pattern, with some myofibers being

normal and some being abnormal. This is subject to sampling error, and again, normal biopsy findings do not exclude carrier status. o Unfortunately, dystrophin immunoblot quantitation, which is very useful in affected males, is not helpful in carrier detection as even female carriers manifesting the disease may have levels within the reference range. If all else fails, linkage analysis comparing polymorphic DNA markers on the X chromosome of an affected patient with those of his mother or sister permits detection of asymptomatic carriers. This can be performed using PCR techniques but requires blood from at least one affected male in the family. On occasion, the results are uninformative (eg, if the mother is homozygous for all markers, discerning which X chromosome harbors the defective gene is impossible). Procedures Muscle biopsy

Despite the specificity of molecular genetic diagnosis, up to 10% of boys with a clinical picture of dystrophinopathies may have no detectable deletions on DNA testing. Therefore, muscle biopsy, while supplanted as the criterion standard, remains an important adjunctive tool, both for quantifying the amount of muscle dystrophin as well as for detecting asymptomatic female carriers. Depending on the purpose of the biopsy, proper site selection is crucial. For detection of female carriers, strong muscles may exhibit no pathology, and very weak muscles may be too devoid of fibers for adequate analysis. For affected males, a very weak muscle may have inadequate tissue for immunoblot and immunofluorescent testing. In addition, the acquisition of muscle tissue from a muscle already severely weak may precipitate further weakness. Therefore, the ideal muscle to biopsy is one that is easily accessible and exhibits moderate weakness (ie, has 80% strength). Two methods are available for assessing dystrophin in muscle tissue. o Immunostaining of the muscle using antibodies directed against the rod domain, carboxyterminals, and amino-terminals of dystrophin protein shows absence of the usual sarcolemmal staining in boys with Duchenne muscular dystrophy. Patients with Becker muscular dystrophy show more fragmented and patchy staining of sarcolemmal regions.

(A) Normal dystrophin staining.(B) Intermediate dystrophin staining in a patient with Becker muscular dystrophy.(C) Absent dystrophin staining in a patient with Duchenne muscular dystrophy. Some consider the most accurate lab method for differentiating Duchenne from Becker muscular

dystrophy to be the immunoblot of muscle homogenates. Patients with Duchenne muscular dystrophy have greatly decreased or absent amounts of truncated dystrophin, whereas patients with Becker muscular dystrophy protein reveal moderately reduced amounts of dystrophin, which may be smaller (ie, deletion of the dystrophin gene) or larger (ie, duplications of the dystrophin gene) than normal. Clinical correlation is more important as there are exceptions to this notion. Histologic Findings Few muscle biopsies are as instantly recognizable as those of patients with Duchenne muscular dystrophy. Features of Duchenne muscular dystrophy are reminiscent of a tissue battlefield after a major conflict, with necrotic muscle fibers littering the landscape. Widespread muscle necrosis leads to angulated fibers, central nuclei, and considerable fiber size variation, with regenerating cells in different stages of atrophy and regrowth. Fibers that are too damaged to regenerate may become empty skeletal remnants or ghost cells. Actively regenerating fibers often display cytoplasmic basophilia, with large nuclei and prominent nucleoli. Damaged fibers exhibit reduced histochemical staining for oxidative enzymes. Initially, macrophages and cluster of differentiation 8-positive (CD8+) T lymphocytes invade necrosing muscle fibers. In time, this cellular response is supplanted by endomysial and perimysial fibrosis and fatty tissue replacement, which convey the macroscopic appearance of pseudohypertrophy. Aside from linkage analysis, fluorescent immunostaining for dystrophin protein can be a way to diagnose carrier status in a family with no known gene deletion or duplication. Antibody staining for portions of the dystrophin molecule at the sarcolemmal membrane reveals the conspicuous absence of various portions of the dystrophin complex. In boys with Duchenne muscular dystrophy, the sarcolemma is virtually devoid of staining (see section C in image below).

(A) Normal dystrophin staining.(B) Intermediate dystrophin staining in a patient with Becker muscular dystrophy.(C) Absent dystrophin staining in a patient with Duchenne muscular dystrophy. In contrast, carrier females exhibit a more variable mosaic pattern consisting of normal and abnormal fibers. Immunoblot analysis of muscle tissue, available through commercial laboratories, can determine the size and quantity of the dystrophin molecule. Patients with Duchenne muscular dystrophy exhibit no dystrophin. In patients with Becker muscular dystrophy, variable amounts of dystrophin are present but with an altered molecular size. Carriers of Duchenne muscular dystrophy exhibit mosaicism for dystrophin expression and usually have enough functional dystrophin to be within normal limits on Western blot testing, making this a generally poor method for carrier detection. Medical Care Therapeutic strategies for the dystrophinopathies can be categorized into 3 groups based on their approach: (1) Supportive pharmacologic therapy, (2) research gene therapy, and (3) research cellular therapy. Gene therapy involves viral, plasmid, and

oligonucleotide-based approaches. Cell therapy uses myoblast and stem cell techniques. The therapeutic strategies are usually applied first to Duchenne muscular dystrophy with the thought that benefits can be extrapolated to Becker muscular dystrophy. The gene and cell approaches are more likely to be curative, but they are still under investigation. Until these molecular therapies become clinically available, supportive therapies can be used to protect muscle mass and function and to help improve quality of life. Supportive therapies While no cure yet exists for Duchenne or Becker muscular dystrophy, medical and supportive treatments can have a positive impact to reduce morbidity, increase quality of life, and prolong lifespan. Please see Treat-NMD recommendations for Standards of care for Duchenne muscular dystrophy. Comprehensive care for boys and young men with dystrophinopathy can be pivotal and some centers offer multidisciplinary (different pediatric specialties) and interdisciplinary (coordinated) approaches. Fragmented and limited care is suboptimal for those touched by dystrophinopathy, especially in transition to adult medical care. Muscular dystrophy is not just a muscle disease. New care guidelines have been published for boys and young men with Duchenne and Becker muscular dystrophy that detail out the multidisciplinary management, including role of corticosteroids, dedicated cardiac [4] surveillance, and respiratory expertise. The one proven medical treatment for Duchenne muscular dystrophy is corticosteroids, which are known to help to protect muscle mass and function and ameliorate some of the secondary aspects of this disease, thus improving quality of life. Independent ambulation may be extended up to 3 years. Inflammation is implicated in the pathogenesis of the dystrophinopathies despite the fact that most biopsies in patients with Duchenne muscular dystrophy do not show inflammatory cells. Corticosteroids have been used for more than 40 years with some success to treat patients with Duchenne muscular dystrophy. The central role of inflammation in the pathogenesis of the dystrophinopathies is suggested by the fact that use of corticosteroids, such as prednisone, results in prolongation of ambulation, maintenance of strength and function, and delay in the development of scoliosis. The side effects are well-known and do temper many clinicians enthusiasm to recommend its use in small children, patients with behavior or learning issues, or any patient for chronic use. A detailed understanding of the mechanism of action for corticosteroids on the body is still a large mystery. To date, corticosteroids are the only medication that has demonstrated a modest benefit in modifying the course of the [5] disease. Clinical improvement is seen as early as 1 month after starting treatment and lasts as long as 3 years. Children who discontinue corticosteroids for various reasons soon revert to natural downward progression of the disease. It is hypothesized that prednisone reduces tissue inflammation, suppresses cytotoxic cells, improves calcium homeostasis, and stimulates myoblasts. One challenge is to know if a young boy with elevated serum CK levels is likely to have Duchenne versus Becker muscular dystrophy and if corticosteroids should be initiated in either situation. The muscle biopsy with protein analysis can be valuable in this determination. Some clinicians recommend waiting to start corticosteroids and others believe if Gowers sign is visible then treatment of a low-dose, intermittent regimen should be initiated. The genotype-phenotype correlations in cases of dystrophinopathy have shown "outliers" or exceptions to the rules. Biopsy protein studies in young boys are thought to provide further evidence to help predict Duchenne versus Becker muscular dystrophy phenotype but that has not been conclusively demonstrated and again, there are a small proportion of outliers.

In retrospective and prospective studies, corticosteroids (prednisone and deflazacort) have been found to be favorably associated with 23 years more of independent ambulation, reduced or delayed need for scoliosis surgery, reduced or stabilized ventricular dysfunction, and improved respiratory function. These associated favorable measures in Duchenne muscular dystrophy certainly implicate the positive benefits of corticosteroids in improving quality of life and reducing morbidity and mortality. However, clearly significant side effects must be addressed and monitored. Unfortunately, chronic daily use of corticosteroids can cause weight gain, cataracts, osteoporosis, hypertension, diabetes, delayed puberty, stunted vertical growth, and behavioral/sleep issues. Alternate-day dosing of prednisone (0.75-1.5 mg/kg/d) may help reduce the risk and severity of these side effects. Currently, an international trial of steroid dosing regimens is evaluating prednisone 0.75 mg/kg/d versus deflazacort 0.9 mg/kg/d versus [6] prednisone 0.75 mg/kg/d (10 days on, followed by 10 days off). Prednisone may also increase the expression of utrophin, a dystrophin homologue, by stimulating the utrophin promoter. Oxandrolone (Anavar or Bonavar) is an anabolic steroid approved for use in Europe but it is not FDA approved in the United States; it has been used in Duchenne muscular dystrophy and may have a more favorable side effect profile including less excess weight gain. Oxandrolone has shown greater promise than other anabolic steroids because of its action not only on androgen receptors but also by antagonizing cortisol binding to glucocorticoid receptors to decrease catabolic pathways. It has been used with success in patients with HIV and burn victims, increasing lean body mass, and it remains onboard for 6 months after cessation of treatment. Additionally, this medication produces only minor androgenic side effects in children. Preliminary clinical testing in patients with Duchenne muscular dystrophy who are receiving daily oxandrolone showed improvement in muscle strength testing but not in functional testing as compared with controls. No significant adverse effects occurred over the 6-month trial. Additionally, an advantage over corticosteroid use may be that the growth of the subjects was not slowed. Controversies still exist with respect to age to start corticosteroids, clinical criteria to start corticosteroids, which corticosteroid, which dose and which regimen (continuous daily or intermittent regimens), and when to discontinue corticosteroids. Immunizations schedule is generally thought to be a reason to hold off initiating corticosteroids until 4 years of age but there is no question that serum CKs are already elevated in the first year of life. A paucity of published rigorous clinical trials clarify these controversies and hopefully in the future such studies will be illuminating. Some clinicians acknowledge that the use of corticosteroids has a valuable role. However, the ideal dosing/regimen may not be universal given a boy's developmental age and may need to be individually tailored to the childs specific needs and negative side effect profile. Corticosteroids are most often given in the morning with breakfast. Sometimes a daily H2 blocker such as ranitidine can be useful for gastritis/ulcer prevention. Families and physicians must be wary of abdominal complaints. In addition to skeletal muscle abnormalities, cardiomyopathy is also [7] a significant problem in individuals with dystrophinopathy. The extent of cardiac involvement and resultant cardiomyopathy is often a significant determinant of clinical status and long-term outcome, [8] especially for the patients with Becker muscular dystrophy. Corticosteroids have been shown to have favorable effects on [9] cardiac function in Duchenne muscular dystrophy. Studies have shown that afterload reduction with ACE inhibitors in patients with and without ventricular dysfunction leads to better preservation of the myocardium and improvement in ventricular function and [8] geometry. Perindopril, an ACE inhibitor, has been reported to have

a positive influence on cardiac function in a cohort of patients in [10] France. In mdx mouse models, angiotensinogen receptor blocking agents may have a favorable role in both skeletal and cardiac muscle function. Clinical trials are warranted. Overzealous exercise or training can speed up muscular dystrophy and gentle sports or activities (eg, swimming and tricycle/bicycles) may be encouraged. With the supervision of an experienced physical therapist, stretching is also important for parents to incorporate into the home regimen. [11] Osteoporosis and fractures are also significant problems. A small case series of 3 boys with Duchenne muscular dystrophy and known osteoporosis were treated for 1 year with weekly alendronate and daily calcium with vitamin D. Dual-energy x-ray absorptiometry was followed from baseline to 6 months and 1 year. This treatment regimen was found to be effective in improving bone mineral density; however, the study did not address the impact of this [12] treatment on the prevention of long-bone or vertebral fractures. Other bone massenhancing drugs may be worthy of further investigation but much research is lacking in this area. A 12-week trial in boys with Duchenne muscular dystrophy with daily administration of the a2-adrenergic agonist, albuterol, showed an increase in muscle strength on knee extension testing, but no significant difference in muscle function. Clinical trials with calcium channel blockers have shown no benefit. However, dantrolene, a medication that prevents calcium release from the sarcoplasmic reticulum, has shown a mild beneficial effect. Other pharmacologic treatments, such as cyclosporine, cytokine modulation with TNF-a, nitrous oxide regulation, and mitogens, are currently being investigated, but current evidence does not show any significant benefit. One study showed that combining prednisone with cyclosporine A, or using cyclosporine A as a monotherapy, while safe and well tolerated, did not show improved [13] muscle strength or functional abilities. Most treatments have not shown a benefit as significant as that of prednisone. Supportive care Supportive care requires a coordinated and multidisciplinary team approach including primary care physician, neurologist, pulmonologist, cardiologist, endocrinologist, physical therapist, orthotist, mobility expert, nutritionist, orthopedic surgeon, social worker, genetic counselor, psychologist/psychiatrist, palliative care team, and school staff (including teacher, counselors, and nurses). Plays a crucial role in maximizing functional status and tone, as well as in delaying wheelchair dependence. o Daily joint-stretching exercises prevent the debilitating onset of contractures. o Night splints can have a favorable influence. o Judicious use of tendon release surgeries may prolong ambulation by as long as 2 years. o Braces, such as ankle-foot orthoses and kneeankle-foot orthoses, can be adjuncts in prolonging the period of mobility and delaying wheelchair dependency. Maintaining the ability to stand, even without mobility, delays the onset of many contractures and scoliosis. This may require elaborate bracing mechanisms and often is poorly tolerated and expensive. Because bracing delays but does not prevent the eventual outcome, this option is less frequently pursued now than in the past. Once wheelchair dependency becomes more prominent, attention shifts to prophylaxis against the deleterious consequences of immobility. o The chair itself must be chosen carefully and customized to the patient's needs.

Strategic cushioning reduces the incidence of pressure sores with attendant skin breakdown, which often occur in the sacral and coccygeal regions. Adaptive devices, such as specially designed wheelchair tables and ball-bearing splints, maximize upper extremity mobility in muscles that cannot resist gravity. [14] Careful monitoring of pulmonary function is necessary. The forced vital capacity (FVC), provides a rational means for deciding when the patient would benefit from assisted ventilation. Insufflator, exsufflator, or cough assist devices, are believed to greatly reduce the risk of pneumonias/hospitalizations and improve pulmonary health. Sleep studies o Continuous positive airway pressure (CPAP) and the more physiologic bilevel positive airway pressure (BiPAP) are the 2 major options in this regard, both of which are minimally invasive and easy to use. o Daily use of incentive spirometer reduces atelectasis and pneumonia. o X-rays are used to monitor spinal curvature because scoliosis adversely affects respiratory capacity. Spinal instrumentation or even fusion may become necessary if serial x-rays reveal worsening of spinal curvature. As the disease continues to progress, more technology for noninvasive ventilatory support and invasive options include tracheostomy with or without mechanical ventilation. Dietary modifications can prevent excessive weight gain with its attendant strain on transfers and pulmonary function. Great interest in nutraceuticals with antioxidant and antifibrotic properties have been highly sought after by Duchenne muscular dystrophy families. The appeal of avoiding FDA regulatory issues in nutraceuticals is limited by the less stringent and relatively unregulated marketplace of dietary supplements where claims/labels are not held to the standards and inspections of FDAapproved drugs. Issues in this realm include impurity of compounds sold as nutraceuticals. Sometimes nutraceutical labels may claim to have an active compound in it that is actually not included, and, in other cases the concern may be what other extra compounds (contaminants) may be included that are not mentioned in the labeling. Ultimately, sensitive yet candid and thorough discussions with patients and their families are important in making decisions about prolonging life while maximizing quality of life. Family support is an important but complex and underappreciated element in any therapeutic strategy. o Psychologists have observed development of an unusually close relationship between mothers and afflicted sons, often at the expense of siblings and spouses. Family counseling, by fostering open communication and addressing unresolved issues of jealousy, guilt, and anger, may improve this social dynamic. o Educating the family about the natural course of the disease and informing them about the

availability of support groups remain important tasks of the neurologist. Parent Project Muscular Dystrophy Muscular Dystrophy Association Treat NMD, Neuromuscular Network BeckerMD.org BrainPOP, Duchenne Muscular Dystrophy Transitional care and special care primary care providers Transitional care is vital so that boys with dystrophinopathy, especially Duchenne muscular dystrophy, will grow up to be men with dedicated medical care as they achieve benefit from comprehensive care. Unfortunately, one challenge will be for adult care providers to take on men with Duchenne or Becker muscular dystrophy as this is historically considered a pediatric disorder and few patients with Duchenne muscular dystrophy survive into adulthood. New comprehensive approaches will continue to improve the natural history of Duchenne muscular dystrophy and improve health for all touched by dystrophinopathies. As with so many other genetic diseases, much hope resides in molecular genetic advancements, and improving treatments will aim to shift this disorder into a chronic disorder instead of a life-limiting one. Research gene therapy Information about Duchenne and Becker clinical trials can be explored by searching for Duchenne or Becker muscular dystrophy on ClinicalTrials.gov for eligibility criteria. One key component to position this field for clinical trials is for individuals with Duchenne or Becker muscular dystrophy to register in patient databases so that streamlined accessibility can better link clinicians, scientists, and patients with research opportunities. Some invaluable Duchenne and Becker muscular dystrophy registries on which individuals may register are as follows. The DuchenneConnect Profile allows all those living with Duchenne or Becker muscular dystrophy to join the DuchenneConnect patient registry, offering them access to information about new treatments and trials, as well as regional and local resources. Registering with DuchenneConnect also connects members to the global international database (TREAT-NMD Neuromuscular Network). The United Dystrophinopathy Project (UDP) Patient Registry directed by Dr. Kevin Flanigan gathers basic information about patients with Duchenne, Becker, and intermediate muscular dystrophy and invites families to join the UDP registry. Registering with UDP also enrolls participants in the global international database (TREATNMD Neuromuscular Network). Action Duchenne, DMD registry For families outside the United States, visit the TREATNMD Neuromuscular Network. The TREAT-NMD Neuromuscular Network (TREAT-NMD) is a registry network focused on advancing diagnosis, care, and treatment for people with neuromuscular diseases around the world. TREAT-NMD maintains data submitted from all member registries. Click on the link to your country's DMD registry and then you will be registered with TREAT-NMD. Having this information in one place should make it easier for researchers to perform clinical trials to study these diseases. Individuals with Duchenne/Becker muscular dystrophy can become a participant of TREAT-NMD by joining their respective national registry. The aim of gene therapy is to deliver DNA encoding dystrophin or other therapeutic genes, such as utrophin, to muscle. This strategy is

complicated because of the enormous size of the dystrophin gene and difficulty engineering an effective delivery system. Currently, the delivery vectors available cannot accommodate the gene in its native form. Functional studies of the gene in mdx mice have shown that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and microdystrophin genes that have the advantage of being within viral/plasmid cloning capacities. These mini- or microdystrophins can be directly inserted into muscle. Use of naked plasmid DNA does not provoke the vigorous antigenic response that viral vectors do. The problem with directly inserting the DNA into muscle is knowing the exact dose to produce a clinical response and having to insert the DNA into several different muscles separately rather than being able to give it systemically. Additionally, evidence shows that the contractile properties of the muscles are not restored despite significant [6] correction of the underlying membrane defect. The first US trial testing the effectiveness of minidystrophins in humans began in late March 2006 at Columbus Children's Hospital in Ohio. "Booster" genes are beginning to be studied to augment the possible therapeutic effect of these mini- or microdystrophins. Dual gene therapy of the small dystrophins with genes that create an environment for muscle growth or regeneration (including insulin growth factor-1 or genes such as follistatin that inhibit the negative muscle growth regulatory factor myostatin) have been shown to protect muscle against contraction-induced injury and to increase [6, 15] muscle mass in animal models, respectively. Additionally, overexpression of the enzyme Galgt2 has been shown in animal models to be useful in maintaining membrane stability by creating a [6] utrophin-glycoprotein complex. Clinical trials are planned to assess the possible effectiveness of these adjunctive treatments. Modification of endogenous dystrophin is another gene therapy technique under investigation. Most mutations in Duchenne muscular dystrophy cause a disruption of the open reading frame during transcription, which effectively aborts translation to a functional dystrophin protein. Several different techniques can be used to re-establish an open reading frame mutant, resulting in a functional dystrophin mRNA. Targeted exon skipping can restore an open reading frame by modulating the splicing of the Duchenne muscular dystrophy gene. In the case of single or multiple deletions and point mutations, a slightly shorter, but in-frame transcript, would be produced by skipping over a particular exon sequence. This therapy may be even more effective in duplications because of the possible generation of a true wild-type dystrophin from skipping 1 or 2 exons. The mechanism of exon skipping is based on the use of antisense oligonucleotides (AO). AO are small synthetic RNA molecules that can bind to specific sequences within the dystrophin pre-mRNA. This technique could possibly benefit 70-80% of patients with Duchenne muscular dystrophy when a comprehensive panel of specific AOs or cocktails of AOs to treat all of the different dystrophin mutations becomes available. Clinical trials are currently [6] underway to evaluate the safety and tolerability of this treatment. Approximately 10% of Duchenne muscular dystrophy cases and most Becker muscular dystrophy cases are caused by nonsense mutations that induce premature stop codons causing premature translational termination. The most promising compounds capable of suppressing premature termination are the aminoglycosides and PTC-124. These compounds induce ribosomes to readthrough premature stop codons, resulting from nonsense mutations, thus, increasing dystrophin protein expression. The oral agent PTC-124 dose/efficiency of upregulated dystrophin protein expression, which may equate to human functional motor benefit, remains to be proven as do any long-term side effects.

Although promising results were achieved in the mdx mice, human trials with gentamicin failed to show an increase in the expression of dystrophin. PTC-124 has been shown to be superior to gentamicin at ribosomal read through in vitro. Currently, a phase 2b efficacy and safety study in 165 subjects is underway to evaluate this [6] compound. Growth factors have also been tried as a strategy to increase protein production in dystrophic muscles. In a clinical trial with 7 patients with Duchenne muscular dystrophy, exogenous growth hormone (GH) produced undesired, catabolic effects likely secondary to a positive nitrogen balance induced by the hormone. While GH has this effect on skeletal muscle, it has been shown to have a potential beneficial effect on Duchenne muscular dystrophy cardiomyopathy. Given these mixed results, the usefulness of GH in treating Duchenne muscular dystrophy remains in doubt. On the other hand, insulinlike growth factor (IGF-1) may be helpful in protecting muscle mass and function. IGF-1 is a positive regulator of muscle growth and has a profound effect on muscle precursor activation and proliferation. Upregulation of IGF-1 in the mdx mouse showed functional improvement, restoration of muscle strength, and reduced fibrosis. While promising, other studies have shown that IGF-1 can play a key role in proliferation and metastasis of cancer cell and also the occurrence of cancer in humans. IGF-1 has not been clinically tested in patients with Duchenne muscular dystrophy, but such trials may be on the near horizon. Inhibition of calcium-dependent proteases (calpains) can also protect muscle mass. It has been long postulated that calcium homeostasis is disrupted in dystrophic muscle. This disruption in calcium homeostasis is caused by the activity of muscle, which can lead to microlesions of the dystrophic membrane, allowing an abnormal calcium influx that could promote cell death by activating proteases. The actions of these proteases can be aborted by calpastatin, an endogenous inhibitor of calpains. The expression of calpastatins can be increased with 2-adrenergic agonists. Regulation of myostatin may also be another alternative to preserving muscle mass and function. Myostatin is a member of the transforming growth factor (TGF), a superfamily of growth/developmental factors, and is a potent, negative regulator of functional muscle mass. Deletions of the myostatin gene cause muscle cell hypertrophy. One case report exists in the literature of a 4 and a half-year-old boy born with no detectable myostatin in his sera. He had unusually large muscle at birth, with no other detectable abnormalities, including cardiac abnormalities. A phase I study with antimyostatin antibodies injected into patients with muscular dystrophy resulted in no improvement in the muscles. Cellular therapies Unfortunately, clinical trials to date have not shown favorable results with the use of myoblast transplantation or stem cell transplantation into patients with Duchenne muscular dystrophy. Myoblasts (normal muscle precursor cells) can be introduced into dystrophic muscles and incorporated into the myofibers but efficiency of transfer and immunorejection remain problematic. The newly formed myofiber can carry a functional form of the dystrophin gene which, with the help of reverse transcriptase, can result in the production of a normal dystrophin protein that can be incorporated into the sarcolemma. Although shown to be promising in the mdx mouse, human trials did not show any objective benefit and levels of expression were low. These same disappointing results also occurred with the use of stem cell transplantation. Currently, neither therapy is recommended for clinical use. Future molecular therapies Given breakthroughs shown in animal models of Duchenne muscular dystrophy (mdx mouse and GRMD dog) and now human Duchenne muscular dystrophy clinical trials, it stands to reason that the

ultimate cure, dystrophin gene replacement/repair will be realized. Scientific challenges to surmount include the following: age to intervene, efficiency of gene repair in high percentage skeletal and cardiac muscle cells, clinical efficacy to functionally normalize a boy with Duchenne muscular dystrophy, immune rejection issues, longterm side-effects, short-term toxicity. Given the time necessary to establish dosing, safety, and efficacy of new molecular medicine techniques for regulatory approvals (ie, FDA), bridging therapies are needed to slow down the pathogenesis of dystrophin-deficiency. Some critical areas to prioritize include attenuating the fibrotic accumulation, maintaining the overall health of affected individuals, using favorable medicines and nutraceuticals, avoiding deleterious medications or regimens. Some in the field believe that a combination treatment or Duchenne muscular dystrophy cocktail will be necessary to offer an optimum multifaceted approach to slow down muscular dystrophy. Consultations Psychologists Genetic counseling o Genetic counseling remains the sole intervention for preventing the disease. o Initiate genetic counseling soon after the diagnosis has been made. o Maternal genetic testing can assess whether the mother is a carrier (carrier state conveys a 50% risk for any future male progeny) or whether the patient's disease arose from a de novo mutation, which occurs about 30% of the time. o While major dystrophin deletions can be detected in female carriers, linkage analysis occasionally becomes necessary in cases of more subtle point mutations to prove that mother and son share the same X chromosome. o Chorionic villus sampling and amniotic cell analysis permit prenatal diagnosis either by testing for a known deletion or duplication, or by linkage analysis. These procedures should be performed only after extensive counseling that involves discussing the implications of a positive test result as well as the available options. Medication Summary To date, prednisone is the only medication that has demonstrated even a modest benefit in modifying the course of the disease. Clinical improvement is seen as early as 1 month after starting treatment and lasts as long as 3 years. Children who discontinue steroids for various reasons soon revert to natural downward progression of the disease. Corticosteroids Class Summary These agents have anti-inflammatory properties and cause profound and varied metabolic effects. Corticosteroids modify the body's immune response to diverse stimuli. View full drug information Prednisone (Deltasone, Orasone, Meticorten, Sterapred) Anti-inflammatory drug with unknown mechanism of action.