Current Status of Duchenne Muscular Dystrophy

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PEDIATRIC NEUROLOGY

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CURRENT STATUS OF DUCHENNE MUSCULAR DYSTROPHY Susan T. Iannaccone, MD, FAAN

Duchenne muscular dystrophy (DMD) is the second most common lethal genetic disorder in humans, affecting 1 in 3300 live male births.27 Descriptions by neurologists and pediatricians during the mid to late nineteenth century included nearly everything scientists knew about DMD until 1987. For example, Gowers described in his textbook a disorder". . . characterized by a progressive change in the size and diminution in the power, of many muscles."33 He attributed the recognition of this disease as a distinct entity to Duchenne, who called it "pseudo-hypertrophic muscular paralysis" in 1868. 25 Although the preponderance for boys was well recognized, girls were described as having a mild form of the same disease. One of Gowers' female patients was teased for having" 'tea-kettle calves' "33; apparently, she was a manifesting carrier. 70 He, of course, is best known for his exquisite description of " . . . the greatest defect . . . in the power of rising from the floor . . . . "33 His line drawings of the Gowers' maneuver rival Dubowitz' color photographs 2' (Fig. 1). Equally impressive is the fact that Duchenne performed needle muscle biopsies with an instrument almost identical to one used today for diagnosis 56 (Fig. 2). The discovery in 1959 that serum creatine kinase (CK) levels were markedly elevated in young boys with DMD was a major breakthrough. 26 This discovery meant that children with motor delay or other signs of weakness could be screened easily with a blood test. Serum CK was also used to screen women at risk for carrier state. 34 72 If the level was normal, the number of affected and unaffected male relatives was used to calculate mathematically the woman's risk of being a carrier. Although several clinicians suggested using the CK as a routine neonatal screening test, this has never been implemented. Now that From the University of Texas Southwestern Medical School at Dallas; and Neuromuscular Disease and Neurorehabilitation, Texas Scottish Rite Hospital for Children, Dallas, Texas

PEDIATRIC CLINICS OF NORTH AMERICA VOLUME 39· NUMBER 4 • AUGUST 1992

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Figure 1. Gowers' sign . A, The patient rolls onto his hands and knees. B, He attempts to put weight on his feet. C, He is able to bear weight on his legs by finding a wide base and supporting some weight still with his hands. D, Finally, he achieves the upright stance by using his hands to climb up his legs, which are still wide apart.

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Figure 2. Biopsy needle used for percutaneous sampling of muscle. The needle consists of an outer trochar with a window (inner diameter 4.5 mm) through which muscle protrudes in vivo. A second component consists of a cylinder that fits inside the trochar, with a sharp cutting distal edge and a proximal arm for attachment to suction. A stylet fits inside the cylinder in order to push out any hidden tissue when the needle is withdrawn from the muscle. During the procedure, the outer trochar and the second component are inserted closed into the belly of the muscle. The second component is then withdrawn so that the window is open and suction is applied. The window is closed forcefully so that tissue that extruded through the window will be cut. Finally, the apparatus is withdrawn from the muscle with the window closed.

effective therapy may be available within the decade, neonatal screening for DMD may make more sense than it did previously. Another major discovery was the result of "reverse genetics. "19, 62 The genetic abnormality responsible for DMD was localized to the Xp21 locus in the human genome (Fig. 3) as a result of studying s~veral patients with unusual translocations and DMD.6, 35 The abnormal gene was isolated and cloned. 45 By reconstructing mRNA, a protein, dystrophin, was shown to be the product of the DMD gene. 5, 36, 46 Dystrophin was shown to be absent from the muscle of DMD patients by using antibodies that labeled the protein in situ. 3, 37, 39 The structure of dystrophin is well known, but its function is still speculative. 38, 46, 48, 75 Several forms of DMD caused by deletions of various exons from the dystrophin gene have now been separated from other types of muscular dystrophy.2, IS, 20, 31, SO, 63 Animal models for DMD in which the animal is dystrophin-deficient and has a genetic abnormality analogous to the human deletion include the mdx mouse 30, 65 and the Xmd dog. 18, 71 These animal models will be valuable in evaluating future treatments before clinical trials.

DIAGNOSIS

The diagnosis of DMD should be made on the basis of a history, physical examination, and laboratory data. Usually, a small boy presents with an abnormal gait, waddles (Trendelenburg gait), and falls frequently; he has a high CK level, about 104 times normal. Unfortunately, weakness may be mistaken for clumsiness. The presence of mental retardation (up to 30% of DMD patients) may obscure the fact that the patient has weakness rather than developmental delay, Attention-deficit hyperactivity disorder may be of more concern to his parents than his difficulty climbing stairs. The presence of pseudohypertrophy, especially of the calves, but also of any muscle group (Fig. 4), is a helpful sign of DMD, although it is not pathognomonic. The combination of such physical findings and abnormal CK levels in a boy should be considered indicative of DMD. Before proceeding to muscle biopsy, many neurologists recommend ob-

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Charcot-Marie-Tooth disease

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Duchenne/Becker muscular dystrophy

Kennedy's disease Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease

Emery-Dreifuss muscular dystrophy Myotubular myopathy Fragile X syndrome q Figure 3. Human X chromosome with location of gene for Duchenne muscular dystrophy

in relation to other X-linked disorders and the X-linked forms of Charcot-Marie-Tooth disease.

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Figure 4. Pseudohypertrophy of the vastus lateralis and calf muscles.

taining an electromyogram (EMG). However, not all EMGs are equal." The reliability of this test is determined by the training and experience of the examiner as well as the level of cooperation of the patient. The choice of doing an EMG before the muscle biopsy should depend on the index of suspicion of DMD. If the patient presents as a typical case of DMD, then the EMG may not be necessary. The muscle biopsy remains the single most important test for DMD because our definition of DMD now requires that muscle dystrophin be abnormal or absent. 36 Direct analYSis of muscle tissue is necessary. Routine histochemical studies should be done to assure that the histologic analysis of the muscle is consistent with a diagnosis of muscular dystrophy; that is, one should find evidence of necrosis and regeneration of muscle fibers and proliferation of connective tissuez (see Fig. 7A and B). Dystrophin content in muscle can be determined by performing immunofluorescent staining using antibodies directed against one or more parts of the dystrophin molecule 37 (Figs. 5-7). More commonly, a Western blot of the homogenate of muscle tissue is made and examined for the presence of dystrophin. The Western blot will give an amount and molecular weight of dystrophin (Fig. 8). If dystrophin is absent from the patient's muscle, the patient has DMD. If dystrophin is present in an abnormal

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Figure 5. A and B, This biopsy was taken from a 30-year-old male patient. The man had an elevated CK (3000). The light histology (A) is slightly abnormal because of internal nuclei (arrows) and occasional atrophic fibers (arrowhead). Immunofluorescence (B) for dystrophin was normal, and immunoblot indicated that the dystrophin was of normal size. (A, Hematoxylin and eosin stain; bar=0.8 mm. B, Immunofluorescence; bar=1 .6 mm.) (Courtesy of E.P. Hoffman, PhD.)

amount or is of abnormal molecular weight, the patient may have DMD or Becker's muscular dystrophy (BMD), a mild form of the disease caused by a different deletion of the same gene (Table 1).28 BMD has been defined clinically to include those patients who remain independently ambulatory to age 16 years. 9 The mean age at which DMD patients become wheel-chair dependent is 11 years. Patients may have "mild" DMD or "severe" BMD and exhibit a phenotype wherein they lose ambulation between 12 and 16 years; they have been called "outliers" for research purposes. Now with molecular diagnosis available, we know that a patient with severe BMD may have a course identical to that seen in a patient with typical DMD or, conversely, a patient with "mild" DMD may have a slow course similar to that of a patient with typical BMO. Some correlation exists between the location and size of the deletion and the clinical severity of the disease,so.57 but a deletion has not been detectable by routine methods in 40% to 45% of patients with OMO or BMO.20 New Table 1. CLINICAL CORRELATION TO DYSTROPHIN RESULTS Dystrophin Clinical phenotype DMD

=

Absent DMD

Duchenne muscular dystrophy, BMD

Abnormal DMD or BMD =

Becker muscular dystrophy.

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Figure 6. A and B, A biopsy from a 33-year-old man whose CK was 2570. His clinical presentation was consistent with BMD. The light histology (A) shows many features of an active myopathy or dystrophy, including internal nuclei, rounded fibers surrounded with collagen, many split fibers (arrows), and a reactive monocytic infiltration (arrowheads) . Immunofluorescent stain for dystrophin (B) shows decreased labeling, whereas the immunob lot indicated the presence of dystrophin of 380 kD molecular weight decreased to 60% of normal. (A, Hematoxylin and eosin ; 1 cm = 167 /Lm. B, Immunofluorescent stain; 1 cm= 167 /Lm). (Courtesy of E.P. Hoffman, PhD.)

technology will soon make identification of point mutations possible (P. R. Clemens, MD, et al, personal communication, 1992). It is important to remember that an autosomal recessive form of muscular dystrophy63 shares an identical phenotype with DMD/BMD. SJ In this disease, which affects boys and girls equally, dystrophin is normal, although it has been proposed that a dystrophin-related protein (DRP) may be abnormal. 44, 49

MANAGEMENT Once the diagnosis of DMD/BMD has been confirmed, the physiCian should review test results thoroughly with the patient's parents. Time should be allotted to review the patient's prognosis, the plan of management, and the mode of inheritance. The plan of management should address the main complications of muscular dystrophy: orthopedic deformities, respiratory insufficiency, cardiac failure, and psychological needs, induding both learning and behavior problems (Table 2). Orthopedic complications of DMD tend to occur early, especially flexion contracture in the lower extremities. The mechanism whereby these contractures arise has been somewhat controversial. One theory suggested that

Figure 7. A-C, A biopsy from a 9-year-old boy with clinical DMD. He had a positive family history and was nonambulatory secondary to progressive weakness. Hematoxylin-eosin stained sections showed a marked increase in endomysial connective tissue, variability in fiber diameter, and large hypertrophic fibers (A). Staining with alkaline phosphatase was positive (B, arrowheads), indicating the presence of regenerating fibers. (A and B, magnification = 330 x. ) The light histology of DMD and BMD are quite similar, whereas the immunofluorescence is not. This biopsy (C) is from a patient with confirmed DMD. As is typically seen, there is no subsarcolemmal labeling for dystrophin. Absence of dystrophin was confirmed by immunoblot. (Bar= 1.6 mm.) (Courtesy of E.P. Hoffman, PhD.)

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Figure 8. Immunoblot of muscle homogenate compares normal dystrophin (arrow) to abnormal protein. Patient 1: 47-year-old man carried a clinical diagnosis of BMD, but the dystrophin was normal. Patient 2: 7-year-old boy had a clinical diagnosis of BMD. Dystrophin was decreased in amount and molecular weight and correlated with a gene deletion. Patient 3: This was a 14-year-old male patient. Because he was still ambulatory, he was believed to be an "outlier." Dystrophin was absent, making the diagnosis DMD. Patient 4: 11-yearold boy was believed to have BMD clinically. Dystrophin was markedly decreased (15%) but of normal molecular weight (400 kD), indicating either BMD or DMD. (Courtesy of E.P. Hoffman, PhD.)

contractures were the result of proliferation of fibrofatty tissue such as is seen in muscle biopsy material. Another theory is that flexion at the hip and knee occurs early so that a balanced posture will compensate for weakness and causes toe walking, which results in shortening of the Achilles tendon. Toe walking is frequently the patient's chief complaint. The physical therapist should participate as part of the management team from the time of diagnosis. The therapist should introduce the family to passive range of motion exercises and show them how to do the exercises with instructions to perform them at home daily. Parents should take primary responsibility for this aspect of care, although it is clear that contractures may occur despite the most rigorous of programs. 73 Before considering surgical correction of deformities, goals must be Table 2. COMPLICATIONS OF DUCHENNE MUSCULAR DYSTROPHY AND BECKER'S MUSCULAR DYSTROPHY Orthopedic deformity Flexion contractu res Scoliosis Cardiorespiratory complications Hypoventilation Respiratory failure Cardiomyopathy with congestive heart failure

Psychosocial complications Mental retardation Marital discord Depression Financial burden

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well defined. 53 The patient's mobility and quality of life should remain the most important goal. Scoliosis may appear while the patient remains ambulatory, but it rarely progresses until the patient has become wheelchair dependent. 67 Most specialists now recommend the correction of scoliosis in patients whose curve is greater than 40%.64 The timing of back surgery in DMD depends on the overall health of the patient, severity of curve, and respiratory status or risk of anesthesia. Whether this surgery gives the patient a longer life span is not clear. Pulmonary function tests changed significantly after surgery in one group of dystrophy patients. 47 Some discrepancy exists between results of evaluations by orthopedists and those by neurologists, 54, 69 the latter having shown in agematched populations that correction of scoliosis had no effect on pulmonary function tests. However, the neurologists were able to show an improved quality of life. Correction of scoliosis seems to prevent severe pain because a progressive deformity results in an inability to position comfortably. Several reports of malignant hyperthermia in DMD/BMD patients have appeared. 13 A definite increased risk of rhabdomyolysis and myoglobinuria during anesthesia occurs in patients with muscular dystrophy. The presence of cardiomyopathy also means that sudden arrhythmia or asystole may occur during anesthesia. Therefore, appropriate precautions should be taken by the anesthesiologist before surgery, and the family should be advised of the risk of such events. As muscle weakness progresses in DMD, it involves the truncal muscles and causes increasing respiratory insufficiency. Pulmonary function should be monitored on an annual basis, more often as it decreases. A simple explanation to the parents regarding the mechanism behind the pulmonary difficulties may go a long way toward increasing their motivation in doing chest physiotherapy. All respiratory complications of DMD are mechanical in nature. The immune system and lung tissue are normal. However, a weak cough, dysphagia, and hypoventilation all contribute to increased frequency of pneumonia and, ultimately, respiratory failure. Therefore, mechanical techniques for clearing the airway and improving ventilation are indicated. Bronchodilators may improve drainage after chest percussion. Intermittent positive pressure breathing may reverse atelectasis. Negative or positive pressure ventilation during sleep may improve oxygenation and endurance during the day. 17,58 I generally recommend the vaccines against pneumococcal pneumonia and influenza to decrease the probability of infection. Prophylactic antibiotics introduce the risk of contracting a resistant organism and are not recommended. The concept of respiratory therapy should be introduced at the time of diagnosis. 68 The therapist can instruct the parents in chest percussion and assisted cough early and later hold follow-up sessions to discuss other techniques as they are indicated by the child's condition. Although cardiac muscle is histologically abnormal in most DMD patients, only about 10% develop symptoms from their cardiomyopathy.28 Dystrophin is present in normal cardiac muscle3, 14 and is thought to be abnormal in DMDI BMD, but this hypothesis has not yet been proven. Those patients who become symptomatic do so in early to mid adolescence and usually die of congestive heart failure within 2 to 3 years of onset of symptoms. Thus, this subgroup has a markedly shortened life span relative to the majority of DMD patients. It is beneficial to discuss the respiratory and cardiac complications of muscular dystrophy once the diagnosis is established, and, after the family has had some time to digest the information, one should discuss whether they and the patient would like to use artificial ventilation. The goal should be to arrive at a plan

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that is satisfactory to the patient and his family and to make the plan clear to all caretakers. It can be placed in the patient's chart in the form of a letter or a "living will." Hurried decisions in the heat of a crisis are frequently regretted later and place an unfair burden on emergency room physicians and family members. Several aspects of DMD require the attention of a child psychologist or psychiatrist. Psychometric testing should be performed to establish the patient's intellectual capabilities. This information will aid in planning the child's educational program. Because the patient has a significant physical handicap and can expect to live well into the third decade of life, he or she will benefit from having the ability to support himself or herself in a job or career without physical demands. This means that education will be important for long-term planning. If the patient does have attention-deficit hyperactivity disorder, medication may improve school performance and behavior. Most families benefit from some counseling during the grieving phase after the diagnosis has first been discussed. As is common when chronic disease affects a family member, the frequency of divorce among parents of DMD patients is higher than the national average. It is not rare for the child's father to blame the mother for carrying the "bad" gene. Family counseling should be recommended if symptoms of such dysfunction occur. A combination of family discord, loss of function, and fear of dying nearly always results in clinical depression in DMD patients sometime during adolescence, occasionally sooner. Symptoms of depression are an indication for referral to a specialist. Family and patient support groups sponsored by the Muscular Dystrophy Association are popular with parents and siblings, offering them the comfort of sharing experiences and advice with other people. However, individuals differ in terms of when they are ready to join such groups; some are never willing to participate and they should not be forced to do so. The last area to be addressed by the physician-in-charge is genetic counseling. When the diagnosis of DMDIBMD has been confirmed by dystrophin analysis, then the patient's blood should be examined for a deletion at Xp21. '6 . 19. J5 Deletion detection can now be obtained through several commercial genetic laboratories. Identification of a deletion makes the diagnosis of carrier state in female relatives at risk straightforward, because only those women at risk need undergo phlebotomy. If the patient does not have an identifiable deletion, then the family may want to pursue linkage analysis. Blood samples from all first degree relatives and grandparents of the patient may provide enough information to determine who shares the patient's X chromosome. Girls and women with the same X chromosome as the patient have a mathematically predictable risk of being carriers for the diseased gene. The results and implications of such testing should be reviewed carefully with the family by a trained genetic counselor.

TREATMENT Before 1987, discussions of DMD did not include "treatment." At that time, results of a clinical trial of prednisone indicated that progression of weakness could be slowed so that treated patients did not become wheelchair bound 'at an expected age. 8, 21 Earlier workers had suggested strikingly different conclusions, but they had utilized less convincing statistical methods. 66 The Clinical Investigation of Duchenne Dystrophy (CIDD) group was formed in the 1970s specifically to test the effectiveness of medication in the treatment of

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DMD.9 It was a multicenter clinical collaborative study so that enough patients could be recruited for trials to assure the appropriate "power" of the statistical results.' The investigators tested several "candidate drugs" before prednisone,'2 finding in every case no effect until they used high-dose prednisone. Treated patients became wheelchair-bound an average of 2 years later than untreated patients. The initial trial was followed by several in which the dose of prednisone was lowered, given every other day, or given in a double-blind fashion. 12.29.32 Results of this research are still under discussion for the following reasons:

1. The side effects of prednisone make a true "double blind" study virtually impossible. 2. The side effects of steroids indicate that lower doses of prednisone would be safer. 3. The optimum age at which prednisone should be started is not known, although it is clear that the treatment offers no benefit once the patient has become wheelchair dependent. Because this work is ongoing, it is recommended that DMD patients not receive steroid therapy except under experimental conditions. la, 23 Myoblast transfer is another experimental treatment for DMD. The hypothesis for this work was that transplanted normal muscle cells would fuse with the patient's abnormal cells and that the normal gene for dystrophin would override the defective gene so that the fused cells made normal dystrophin. 40 • 41 Furthermore, it was hypothesized that new production of dystrophin would result in reversal of weakness. Step one was demonstrated in the mdx mouse42• 43. 55 in which normal donor myoblasts were shown to fuse with the recipient's cells. Step two was also completed when the recipient muscle developed increasing levels of dystrophin several weeks following transfer. 60 Step three could not be determined in the mouse. In humans (Fig. 9), what has been shown is that myoblasts from the patient's father fuse with those of the patient to some extent, but steps two and three are still under evaluation. 59 The disadvantages of the method are that it is painful because several injections of myoblasts are required for treatment and repeated biopsies of the treated muscle are necessary to evaluate the result. Moreover, the patient must take cyclosporin to prevent rejection of the transplanted cells. An alternative may be genetic treatment of the patient's own myoblasts followed by performance of an autograft. Attempts to culture myoblasts from young DMD patients and to treat those cells in vitro with transcripts of the dystrophin gene are underway (c. T. Caskey, MD, personal communication, 1992). Precise mechanisms for introducing a normal, artificial gene have not been fully defined. The goal is to provide the patient with his own cells that have been modified to produce normal dystrophin. Whether those cells would fuse with untreated cells after injection and produce dystrophin in vivo is not known. Genetically modified myoblasts have been used in an animal model to treat growth hormone deficiency.4.22 Direct injection of genetic material into muscle has been proposed as gene therapy for DMD. Wolff and his colleagues have performed similar experiments in mice using genes for enzymes that could be identified colorimetrically.74 This work proved that naked DNA could enter muscle cells even at some distance from the injection site and that this DNA could become active, producing a correct protein gene product. One important obstacle for similar feats with the dystrophin gene is its size; it is the largest human gene known to date. Moreover, it is now clear that in a multinucleated cell like a muscle fiber,

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Figure 9. Procedures used for myoblast transfer therapy in human DMD subjects.59 Muscle biopsy of a genetically normal first degree relative, the father, provided a source of muscle precursor cells. The myoblasts were cultivated in tissue culture and then harvested. A cell suspension of myoblasts was injected at intervals into the patient's biceps muscle. The patient was treated with cyclosporin to prevent rejection of the transferred cells secondary to incompatibility of the class 1 major histocompatibility complexes. Results of myoblast injection were evaluated by measuring muscle strength and production of dystrophin in tissue from the treated biceps muscle.

nuclear domains exist for individual nuclei. Thus, dystrophin may appear in a segmental fashion if some nuclei contain the normal gene whereas others do not. So, not only must the genetic material somehow enter every muscle fiber, but also every nucleus, in order to be effective in reversing the dystrophic process. Several possible viral vectors that may be used to overcome these obstacles are under investigation. '. 61 ACKNOWLEDGMENT The author would like to thank Pat Rogers, Media Productions, Texas Scottish Rite Hospital for Children, for preparing the illustrations for the manuscript, and Eric Hoffman, PhD, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, for contributing the materials demonstrating normal and abnormal dystrophin.

References 1. Acsadi G, Dickson G, Love DR, et al: Human dystrophin expression in mdx mice

after intramuscular injection of DNA constructs. Nature 352:815, 1991 2. Arahata K, Hoffman EP, Kunkel LM, et al: Dystrophin diagnosis: Comparison of dystrophin abnormalities by immunofluorescence and immunoblot analyses. Proc Nat! Acad Sci 86:7154, 1989 3. Arahata K, Ishiura S, Ishiguro T, et al: lmmunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature 333:861, 1988 4. Barr E, Leiden JM: Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 254:1507, 1991

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5. Beam KG: Localizing the gene product, Duchenne muscular dystrophy. Nature 333:798, 1988 6. Bodrug SE, Ray PN, Gonzales IL, et al: Molecular analysis of a constitutional Xautosome translocation in a female with muscular dystrophy. Science 237:1620, 1987 7. Brooke M, Fenichel GM, Griggs RC et al: Clinical investigation in Duchenne dystrophy: 2. Determination of the "power" of therapeutic trials based on the natural history. Muscle Nerve 6:91, 1983 8. Brooke MH, Fenichel GM, Griggs RC, et al: Clinical investigation of Duchenne muscular dystrophy: Interesting results in a trial of prednisone. Arch Neurol 44:812, 1987 9. Brooke MH, Griggs RC Mendell JR, et al: Clinical trial in Duchenne dystrophy. 1. The design of the protocol. Muscle Nerve 4:186, 1981 10. Brown RH: Editorial: Prednisone therapy for Duchenne's muscular dystrophy. N Engl J Med 320:1621, 1989 11. Buchthal F, Kamieniecka Z: The diagnostic yield of quantified electromyography and quantified muscle biopsy in neuromuscular disorders. Muscle Nerve 5:265, 1982 12. Burrow KL, Coovert DO, Klein CJ, et al: Dystrophin expression and somatic reversion in prednisone-treated and untreated Duchenne dystrophy. Neurology 41:661, 1991 13. Bush A, Dubowitz V: Fatal rhabdomyolysis complicating general anaesthesia in a child with Becker muscular dystrophy. Neuromuscular Disorders 1:201, 1991 14. Chelly J, Kaplan J-C Maire P, et al: TranSCription of the dystrophin gene in human muscle and non-muscle tissues. Nature 333:858, 1988 15. Clarke A, Davies KE, Gardner-Medwin 0, et al: Xp21 DNA probe in diagnosis of muscular dystrophy and spinal muscular atrophy. Lancet 1:443, 1989 16. Clemens PR, Fenwick RG, Chamberlain JS, et al: Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms. Am J Hum Genet 49:951, 1991 17. Colbert AP, Schock NC: Respirator use in progressive neuromuscular diseases. Arch Phys Med Rehabil 66:760, 1985 18. Cooper BI, Winand NI, Stedman H, et al: The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs. Nature 334:154, 1988 19. Darras BT: Molecular genetics of Duchenne and Becker muscular dystrophy. J Pediatr 117:1, 1990 20. DenDunnen IT, Grootscholten PM, Bakker E, et al: Topography of the Duchenne muscular dystrophy (DMD) gene: F1GE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 45:835, 1989 21. DeSilva S, Drachman DB, MelIits 0, et al: Prednisone treatment in Duchenne muscular dystrophy: Long-term benefit. Arch Neurol 44:818, 1987 22. Dhawan J, Pan LC Pavlath GK, et al: Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 254:1509, 1991 23. Dubowitz V: Editorial: Prednisone in Duchenne dystrophy. Neuromuscular Disorders 1:161, 1991 24. Dubowitz V: Color Atlas of Muscle Disorders in Childhood. Chicago, Year Book, 1992 25. Duchenne de Boulogne GB: Recherches sur la paralysie musculaire pseudo-hypertrophique, ou paralysie myo-sclerosique. Archives of General Medicine 11:5, 1868 26. Ebashi S, Toyokura Y, Momoi H, et al: High creatine phosphokinase activity of sera of progressive muscular dystrophy. J Biochem 46:103, 1959 27. Emery AE: Population frequencies of inherited neuromuscular diseases-a world survey. Neuromuscular Disorders 1:19, 1991 28. Engel AG: Duchenne dystrophy. In Banker BQ, Engel AG (eds): Myology. New York, McGraw Hill, 1986, P 1185 29. Fenichel GM, Mendell JR, Moxley RT, et al: A comparison of daily and alternate-day prednisone therapy in the treatment of Duchenne muscular dystrophy. Arch Neurol 48:575, 1991 30. Geissinger HD, Rao PV, McDonald-Taylor, CK: "mdx" mouse myopathy: Histopathological, morphometric and histochemical observations on young mice. J Comp Pathol 102:249, 1990 31. Gillard EF, Chamberlain JS, Murphy EG, et al: Molecular and phenotypic analysis of

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Susan T. Iannaccone, MD, FAAN Texas Scottish Rite Hospital for Children 2222 Welborn Street Dallas, TX 75219