Chapter 21 Dystrophinopathies

Chapter 21 Dystrophinopathies

Clinical Neurophysiology of Disorders of Muscle and Neuromuscular Junction. Including Fatigue Handbook of Clinical Neurophysiology, Vol. 2 Erik Stalbe...

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Clinical Neurophysiology of Disorders of Muscle and Neuromuscular Junction. Including Fatigue Handbook of Clinical Neurophysiology, Vol. 2 Erik Stalberg (Ed.) © 2003 Elsevier B.Y. All rights reserved

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CHAPTER 21

Dystrophinopathies Karin Edebol Eeg-Olofsson* Department of Neuroscience. Section for Clinical Neurophysiology. University Hospital. S-75I 85 Uppsala, Sweden

21.1. Introduction Since Duchenne de Boulogne (1868) described a pseudohypertrophic form of muscular dystrophy mainly affecting boys, later designed as a dystrophinopathy, science has in fascinating detail shown the molecular basis for this and other forms of muscular dystrophies. Becker and Keiner (1955) described an X -chromosomal muscular dystrophy, similar to Duchenne's muscular dystrophy (DMD), but with milder clinical phenotype, called Becker's muscular dystrophy (BMD).

21.1.1. Genetic background to DMD and BMD Both DMD and BMD are caused by defects in the same gene (Monaco and Kunkel, 1987). The disorders are inherited as X-linked recessive conditions. The gene location of dystrophin is on the short arm of the X chromosome in the band Xp 21.3-p21.2. The gene product, i.e. dystrophin bears the symbol DMD (DYS). The gene is extremely large, and encompasses almost O. I % of the total human genome (Koenig et aI., 1987). However, just a small part of the total genomic RNA, 14 kilobases, is transcribed into a 427 kiloDalton (kDa) protein, which is absent or much reduced in DMD, and reduced in BMD (Hoffman et aI., 1987). The dystrophin gene is expressed as isofonns in skeletal, cardiac and smooth muscles (Dp427m). It is also as in the central nervous system (CNS) in the cortex and hippocampus (Dp427c), and in the Purkinje cells of cerebellum (Dp 427p). It is also expressed in retina (Dp260), kidney, CNS (DpI40), the Schwann cell membrane of adult peripheral nerves (Dp116), and with smaller isofonns (Dp71 and Dp40) in

* E-mail address:

[email protected] Tel.: +46 18611 3447; fax: +46 18556106

various tissues. The dystrophin gene contains 79 exons in the 427 dDa isofonn. In skeletal and cardiac muscles the dystrophin protein is located in the subsarcolemma. In DMD, no functional protein is produced, while in BMD the dystrophin protein is abnormal and only partially functional (Arahata et aI., 1989). The dystrophin protein belongs to a complex, the dystrophinglucoprotein complex, which forms a structural linkage between the cytoskeleton and the extracellular matrix of the muscle fibers. The proteins in this complex are organized into three subcomplexes (Cohn and Campbell, 2000), where the dystrophin and syntrophin proteins constitute the cytoskelatal proteins. This cytoskeleton is connected to the next subunit, i.e. the sarcolemmal localized dystroglycans (alfa and beta subunits) and the sarcoglycans (alfa, beta, gamma and delta subunits) and sarcospan (Crosbie et al., 1997). This subunit in turn connects to the extracellular matrix with the laminin alfa2 protein. Abnormalities in the components of the subunits are responsible for different forms of muscular dystrophies, since the role of the dystrophin-glycoprotein complex is to provide mechanical support to the plasma membrane during muscle fiber contraction. Thus, complete loss of dystrophin as in DMD disturbs the dystrophin-glycoprotein complex and leads to a reduction of the dystroglycans as well as the sarcoglycans and sarcospan (Crosbie et al., 1999). The dystrophin interacts with the other subcomplexes through its carboxyl (COO H) terminal domain where it directly binds to the COOH terminal (C-terminus) of beta-dystroglycan. The other end of dystrophin has a NH 2 terminal (Nterminus) which interacts with filamentous actin to many actin side-binding proteins. By basic repeats in the dystrophin rod domain, F-actin is bound through an electrostatic interaction (Amann et al., 1998). Deletions, involving one or more exons of the dystrophin gene, account for about 65% of DMD

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(Koenig et aI., 1987). Around 6% of DMD patients have duplications (Hu et al., 1990), and in about 30% there are mutations of varying kinds, such as small deletions or insertions, single base changes, or splicing mutations of the dystrophin gene (Prior et aI., 1995). Deletions in BMD are also by far the most frequent type of mutations. Most of DMD patients have a frame-shifting mutation with inability to produce dystrophin beyond next stop codon; the carboxy terminus can then not bind to the dystrophin associated protein complex, and dystrophin becomes rapidly degraded. BMD usually has an in-frame mutation; there is a loss of sequences in the rod domain of the protein leaving N- and C-terminals intact. Mutations near the the N terminal give a more severe form of BMD; N-terminal actin binding region up to the two first rod domain repeats being considered essential for dystrophin function (Anderson, 2002).

21.2. Clinical features 21.2.1. DMD boys 21.2.1.1. Symptom progression DMD occurs in 1: 3500 male births. The onset is in early childhood, usually at 3-4 years. The disease is slowly progressive in young children and thus it may be overlooked in the early stages. Independent walking may be achieved within normal limits or a little later but is abnormal from the onset. The gait is slow, the child never learns to run in a normal way and has difficulties in climbing stairs. The gait is waddling and the child leans sideways when walking in order to compensate for a tendency to fall to the side where the foot is off the ground. Weakness of the hip extensors leads to the development of a pronounced lumbar lordosis. The boy stands with the feet apart in order to increase stability. There is difficulty to raise from a chair or from the floor. In 1878 Gowers had collected 220 cases and gave an illustrated description of the maneuver the patients had adopted when rising from the floor and "climbing up their own legs" (Gowers, 1879). Enlargement of muscles due to muscular fibrosis and fatty infiltration, especially of the calf muscles, has rendered the name pseudohypertrophic muscular dystrophy. The muscle pseudohypertrophy increases with age and is seen in over 90% of the cases. The

K.E. EEG-OLOFSSON

muscular weakness is more pronounced in proximal muscles. The Trendelenburg sign may be seen as the boy stands on one leg causing the hip to dip down on the side where the foot is raised. The muscle weakness is symmetric in upper and lower extremities, but there is a selectivity of muscle involvement. In the upper limbs, the latissimus dorsi, serrati, sternocostal head of pectoralis major, biceps, triceps and brachioradialis muscles are particularly affected, whereas in the lower limbs the quadriceps, iliopsoas and glutei muscles are more involved (Kakulas et aI., 1999). The stretch reflexes are usually depressed, with the exception of the ankle jerks, which may be preserved even in late stages of DMD. During the ages 6-11 years there is a steady decline in muscle function. Contracture of the achilles tendons is common and the child becomes a tip toe-walker. Contractures are also common in the knees and hips. Immobilization in bed in connection with disease or surgery may lead to rapid progress of contractures. Patients with typical DMD become wheelchairbound by the age of 12 years (9-13 yrs). However, steroid treatment may in some boys prolong ambulation by 2-3 years. After loss of ambulation, scoliosis develops in the vast majority and may be rapidly progressive. Cardiac involvement may be present, in early stages only as electrocardiographic changes, but later cardiac incompensation may develop. Respiratory failure occurs, partly due to weakness of respiratory muscles and partly because of the scoliosis. The prognosis is fatal with death occurring between 15 and 25 years of age due to respiratory or cardiac failure. "It is a disease of early life and of early growth. Manifesting itself commonly at the transition from infancy to childhood, it develops with the child's development, grows with his growth - so that every increase in stature means an increase in weakness, and each year takes him a step further on the road to helpless infirmity, and in most cases to an early and inevitable death" (Gowers, 1879).

21.2.1.2. Brainfunction in DMD boys Intellectual impairment is a part of the spectrum of DMD. Also Duchenne himself noted that some boys had a cognitive deficit (Duchenne, 1868). The mean IQ is 85, but there is a great variability. The mental impairment is not related to severity or duration of the disorder. Around 20--30% of the patients have an IQ below 70 (Dubowitz, 1989b), In a recent study on 80 boys with DMD (Hinton et aI.,

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DYSTROPHINOPATHIES

2000) a specific cognitive profile was reported, namely poor performance in digit span, story recall and comprehension. There is a great individual variation in cognitive functions, possibly due to different gene mutations. As to a genotype/phenotype correlation in cognitive dysfunction in DMD, there are so far no conclusive results. One study has shown point mutations in the carboxy terminus of dystrophin in DMD patients with mental retardation (Lenk et al., 1993), and another study revealed that the degree of cognitive dysfunction was related to the presence of mutation in the Dp71 isoform of dystrophin (Moizard et al., 2000). The cognitive deficits do not seem to be progressive (Cotton et al., 1998). In a recent study (Wicksell et al., 2002, submitted to DMCN) 20 DMD boys were compared to 17 normal controls. There was no significant difference in intellectual ability (IQ) between the groups. However, the DMD boys performed significantly poorer on all aspects of memory as well as on new learning and executive functions. In search of gross or histological abnormality of the brain in DMD boys, one study reported that only three out of 21 DMD boys showed abnormalities (Dubowitz and Creme, 1969). On the other hand, slight to severe brain abnormalities in DMD patients, such as neuronal loss, heterotopias, gliosis, Purkinje cell loss and dendritic abnormalities have been reported (Rosman and Kakulas, 1966; Jagadha and Becker, 1988). In a study on 30 DMD patients, a slight cortical atrophy was found in 67%, and this was more common in patients older than 10 years (Yoshioka et al., 1970). Slight cortical atrophy was also found in 60% of 15 DMD boys, especially those with a lower IQ and older age (Septien et al., 1991). However, the correlation between brain abnormality and cognitive impairment has still to be clearly outlined. Biochemical lesions have been reported in cerebellar and hippocampal areas. This is of some interest since there is normally a high expression of dystrophin in the neurons of these regions (Anderson et al., 2002). Electroencephalographic (EEG) studies on DMD patients have revealed abnormalities (Perstein et al., 1960; Nakao et al., 1968) although the correlation between cognitive function and EEG findings is unclear. A report on transcranial magnetic stimulation in four DMD boys has shown reduced cortical excitability compared with controls (Di Lazzaro et al., 1998). Further research on central nervous system dystrophin in DMD will hopefully

give increased understanding of the cognitive problems. 21.2.1.3. Dystrophinopathy in the contiguous gene syndrome A somewhat extended dystrophinopathy is the contiguous gene syndrome located at Xp21 which includes DMD, glycerol kinase deficiency and sometimes adrenal congenital hypoplasia (Darras and Franke, 1988; Seltzer et al., 1989). In a case report (Amato, 2000) the combination of DMD and glycerol kinase deficiency was described in a young boy: at 6 months of age he became stuporous after an upper respiratory tract infection, and at 2 years of age he had a global developmental delay also affecting the speech severely. Serum creatine kinase (CK) was then markedly increased and he had hyperglycerolemia and hyperglyceroluria. Mutation analysis by polymerase chain reaction (Pf'R) revealed a deletion from exons 8 through 79, and fluorescence in-situ hybridization (FISH) showed a deletion involving the glycerol kinase gene. Stupor and global developmental delay are not typical for classic DMD. Symptoms of the associated glycorol kinase deficiency and adrenal congenital hypoplasia in the contiguous gene syndrome may make the diagnosis DMD difficult. Most patients have a DMD phenotype, but a BMD phenotype may also occur (Guggenheim et al., 1980; Towbin et al., 1989). 21.2.2. BMD patients

A milder form of dystrophinopathy is BMD with onset after 5 years of age up to adulthood. It occurs in about 1: 35000 male births, thus constitutes around 10% of the X-linked muscular dystrophies. The disorder is slowly progressive. The severity and age of onset correlate with the muscle dystrophin levels. Most BMD patients are ambulatory after the age of 20. However, some become wheelchair-bound during the adolescence. The disorder is symmetric and predominates in proximal muscles. Often a formal examination in the early stage of the disorder may reveal very little, however the pattern of walking, running and stair-climbing shows a mild pelvic girdle weakness. The hamstrings and quadriceps muscles are usually involved. Enlargement of calf muscles is common. In some, calf pain on exercise is the main complaint. In a study of 67

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BMO patients, 81% had experienced calf pain at some stage of the disease (Bushby and GardnerMedwin, 1993). Muscle cramps are often an early symptom. Contractures are uncommon but may be seen; spinal deformities do not usually occur (Mastaglia and Kakulas, 1996). Cardiac involvement may occur, also before severe weakness is noted, and some patients may require heart transplantation. Cognitive dysfunction is usually not present but may occur in the intermediate forms with lower dystrophin levels. However, BMO may present with a cognitive dysfunction as its major feature (North et aI., 1996). The rate of progression of the disease is variable. BMO is compatible with a normal life span, although some patients may die early. 2/.2.3. Symptomatic female carriers Female carriers are usually not symptomatic but there are unusual cases with symptoms ranging from a Duchenne-like phenotype to mild expression with late onset (16-48 years) and moderate, slowly progressive muscular weakness. Inactivation of Xchromosome has been found in rare cases with a Duchenne-Iike phenotype (Richards et al., 1990; Avner and Heard, 2001). In patients with muscular weakness, involvement of proximal muscles dominates but the distribution is asymmetric, the arms being more involved than the legs. In a recent study on DMOIBMD carriers, 12% had skeletal muscle weakness and 7% cardiomyopathy (Grain et al., 200 I). Some female carriers have symptoms of cramps and myalgia. One report describes a 6-year old female with a sudden and severe episode of rhabdomyolysis (Romero et al., 2001). Muscle biopsy showed a mild necrosis and regeneration, and a mosaic pattern of dystrophin deficiency and in the dystrophin deficient muscle fibers the four proteins of the sarcoglykan complex were lacking. A duplication of exons 3-17 on one X-chromosome of the proband was found but not on the mother's Xchromosome. A skewed X-inactivation was found, consistent with the patient being symptomatic 2/.2.4. X-linked dilated cardiomyopathy Some patients present with X-linked dilated cardiomyopathy (XLOC) as the sole clinical symptom. Patients with this phenotype are usually either

K.E. EEG-OLOFSSON

young males or middle aged female carriers. Whereas the young males present with a lethal congestive heart failure, which is rapidly progressive, the females have a history of atypical chest pain, which will result in a progressive heart failure over years. Muscle biopsies on patients with XLOC show myopathy in skeletal muscles (Mestroni and Giacca, 1997). CK is elevated in most patients, and deletion of the dystrophin muscle-promotor region with abnormal protein expression in heart muscle has been reported (Muntoni et al., 1993). 21.2.5. Atypical dystrophinopathies Asymptomatic individuals with dystrophin gene mutations have been reported (Morrone et aI., 1997) as well as subclinical forms of BMO attributed to the varying age of onset of clinical symptoms (Angelini et al., 1994) of BMO. Some patients with dystrophin gene deletions present with cramps and myoglobinuria in connection (Samaha and Quinlan, 1996). Familial X-linked myalgia and cramps is reported (Gospe et aI., 1989). 21.3. Diagnostic evaluation in dystrophinopathies

21.3./. Clinical signs and symptoms The clinical features constitute the door that opens up for further investigations, and the symptoms determine what a clinician will suspect. 21.3.2. Biochemical tests If DMD or BMO is suspected, an analysis of serum CK is performed. In the early stages of DMD serum CK is very high and increases further with a peak before independent mobility is lost, whereupon it decreases. This relates to the number of dystrophic fibers, which are the source of the elevated CK. As most muscle fibers are lost in the late stages, the serum CK thus is low. The serum CK in OMD is more than 10 and sometimes up to 100 times that of normal. Also all BMO patients have increased serum CK but to a lesser degree, and more than five times the normal value can be seen (Zats et al., 1991). In XLOC, most patients have elevated serum CK, but

DYSTROPHINOPATHIES

normal values have been reported (Mestroni et al., 1999). CK analysis has also been used to detect carriers. 21.3.3. Electrodiagnostic tests 21.3.3.1. Electromyography (EMG) EMG shows myopathic changes. Depending on severity of the involvement at investigation, the MUPs have more or less myopathic features (Figs. 1 and 2), and the EMG is able to quantify the degree of muscle disease. In late stages of DMD the muscle may become fibrotic with areas that are electrically silent. Preferential muscles for EMG are the proximal ones, especially biceps and triceps in the upper extremity, and in the lower limb quadriceps and glutei muscles where more abnormal EMG findings are expected compared to distal muscles. These muscles are easy to examine in children. However, in children who do not cooperate optimally, there is a possibility to do a fairly quick and still reliable EMG with semiquantitative analysis of the MUPs. In an early report (Pascual et al., 1982) 37 males with DMD aged 14 months to 10 years were investigated with EMG. The most frequent findings in EMG were increase of polyphasia and presence of late components in MUPs. Decrease of the duration of MUPs was found only in 50% of studied children. In a study on 14 boys with DMD (Cruz Martinez and Lopez- Terradas, 1992) the MUPs parameters showed a striking increment of long duration MUPs followed by satellites and increase of polyphasic potentials of variable duration. Satellite components were found in 34% of MUPs recorded from 30 patients with DMD (Rowinska-Marcinska et al., 1993), and there was a significant positive correlation between satellite potentials in DMD and muscle force. Complex MUPs were also reported in another study on DMD (Rowinska-Marcinska et al., 1997), and reflect remodelling of the motor unit with muscle fiber diameter variability and distribution of preserved muscle fibers within the studied motor units. In a comparative study on DMD and BMD (lshpekora et al., 1999), 40 BMD patients and 71 DMD patients were investigated with EMG. EMG signs of myopathic changes and spontaneous activity were found in both diseases. Spontaneous activity complex repetitive discharges and fibrillation potentials, as well as positive sharp waves were more

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common in DMD. Measurement of muscle fiber conduction velocity by standard EMG was carried out of 32 patients with DMD in the biceps brachii and anterior tibial muscles (AI-Ani et al., 2001). There was a significant slowing of muscle fiber conduction velocity probable due to the small size of the regenerating and splitting fibers. There was also a multi-peak frequency distribution of the velocities, indicating a great variability in the diameters of the muscle fibers. Reinnervation is preserved in DMD as was described in a patient with an obstetric paresis in his left arm in addition to DMD (Dubrovsky and Taratuto, 1983). EMG showed signs of reinnervation by sprouting superimposed on myopathy in the patient's left arm. Quantitative EMG in 24 familial and sporadic female carriers of DMD and BMD (HausmanovaPetrusewicz et al., 2000) revealed indications of myopathy, and features consistent with dystrophinopathy in various laboratory tests (CK was in all but one female very high, and significantly higher in isolated carriers; immunohisto-chemical evaluation revealed in all females a mosaic pattern of dystrophin staining; quantification of dystrophin showed a reduced abundance with normal vs. abnormal molecular weight; muscle biopsy showed slight to severe dystrophic features).

21.3.3.2. Single fiber EMG (SFEMG)

Studies on muscular studies has revealed increased jitter, especially in DMD (Stalberg, 1976). Severe, moderate as well as slight DMD showed increased jitter and blocking. Increased jitter during voluntary activation may be a sign of enhanced velocity recovery function (VRF), which indicates abnormal muscle membrane function; however also in stimulated SFEMG, where VRF is low, studies showed increased jitter (Stalberg and Trontelj, 1994). Fiber density show highest values in DMD compared to other muscular dystrophies (Stalberg, 1976). The likely explanation for the increased fiber density is grouping of muscle fibers belonging to the same motor unit (MU), not just packing due to atrophy. Increased fiber density indicate a rearrangement of the MU which could for example be due to innervation of a sequestered part of the muscle fiber after a focal lesion, innervation of a regenerating muscle fiber developed from a satellite cell or recordings from a split muscle fiber.

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A

K.E. EEG-OLOFSSON

~~~~~~~ ~f4----. ~~ ~ ~

--v't--

1~r~~~~ ----iJi----

B

-+-A-+--

~

J'..-j'","'--_

c

. - 4 u - - -~ol--'--

---1N---

~'j-t- - -

~ 500 )IV 10 ms Fig. I. Collection of motor unit potentials (MUPs) from the right vastus lateralis muscle in a 2-year girl with normal muscle (A), from the right vastus lateralis muscle in a 2-year old boy with OMD (B), and in the same boy from right anterior tibial muscle (C). MUP amplitudes in A show a mean value of 649 I.LV, mean MUP duration 8,8 ms; in B 120 I.LV (-3.8 SO from normal mean), mean MUP duration 6,8 ms; and in C 204I.LV (-1.75 SO from normal mean), mean MUP duration 5.8 ms.

SFEMG recordings in situ (monopolar stimulation distally and recording proximally) in the biceps brachii muscle on 14 boys with DMD and 20 control children showed a significant slowing of conduction velocity along muscle fibers in the DMD patients

(Cruz Martinez and Lopez- Terradas, 1990), Slowing and large variation in mean fiber conduction velocity (MFCV) was correlated to standard EMG findings of polyphasic MUPs followed by satellites, Satellites might arise from atrophic muscle fibers with slow

DYSTROPHITNOPATfllES

435

4 [pU]

1 0

OJ

4 [pU]

II~. ~

1 0

t

A

A

•p

•p 0

Our r.51

25

0

4 [pU]

W

1 0

9

A

•p

Our r.51

A

25

0

~: LJ Our. (.5]

25

C

B

Fig. 2. Distributions of log amplitudes (Amp) to duration (Our) in relation to normal values in the same muscles as in Figs. lA, 18 and IC, In A (normal muscle) the distribution is well within normal limits; figure B shows a marked left-shift of the values congruent with a myopathic pattern; in figure C there is also a myopathic pattern, although not as severe as in

B.

conduction velocity. The reported method for MFCV in situ is reliable and easy to apply in children. 21.3.3.3. Macro-EMG Macro-EMG was used in investigation on 10 manifesting DMD carriers aged 9 to 52 years and 20 age-matched healthy females (Szmidt-Salkowska et aI., 1999). Macro-EMG revealed myopathic changes in the biceps brachii muscle in all the carriers (whereas standard EMG found myopathic changes in 5110 carriers). Macro-EMG may supply information of early myogenic lesions in muscle. 21.3.3.4. Neurography: motor and sensory Not reported. In DMDIBMD the peripheral nerves are not involved and the neurography is usually normal. However, when the muscle undergoes atrophy the motor neurography may give low distal amplitudes when recordings are made from foot muscles, and in late stages also in hand muscles (author's observations). 21.3.3.5. Autonomic tests One report reveals changes in sympathetic skin response (SSR) in DMD patients compared to controls (Marcello et al., 1995). The study showed significant differences in latency and amplitude of electric type stimuli, and in amplitude values of deep breething - and exogenous startle type stimuli. The alterations in latency and amplitude of the SSR in

the patients could be the consequence of a lack of dystrophin at the level of the sudoriparous myopithelium in analogy to what has been demonstrated in the mdx mouse. 21.3.3.6. EEG EEG is not included among the diagnostic tests for dystrophinopathy. In general, there is a low incidence of EEG abnormalities in DMD. 21.3.4. Muscle biopsy

Clinical suspicion of DMD and BMD together with EMG findings of a muscular dystrophy will promptly lead to a muscle biopsy and genetic testing in order to verify the diagnosis. A report by Muntoni (2001) has the provocative title "Is a muscle biopsy in Duchenne dystrophy really necessary?" The author argues that despite improved sensitivity of DNA testing, there is still a place for muscle biopsy due to the fact that genotyping not necessarily predicts the phenotype, and that muscle biopsy is better correlated to the phenotype. The biopsy can easily be done percutaneously with a conchotome technique, thus avoiding general anesthesia as is needed for an open muscle biopsy. Sedation with midazolam is preferably given to children, usually orally or rectally, shortly prior to the biopsy (Table 1). Muscle histopathology of DMD is in the early stages of the disease and shows non-specific dystrophic changes with increased variation in muscle

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K.E. EEG-OLOFSSON

Table I Dosage schedule for midazolam as sedation in children prior to muscle biopsy and/or EMG. Administration

Dosage (mg/kg)

Max. dosage (mg)

Effect after (min)

Duration (min)

Rectal

0.2-0.3

5

ID-20 4-5 15-30

3D-50 2D-30 3D-50

2-3

15-30

Nasal

0.2

Oral

0.3-0.5

5 7.5

Intraveneous

0.1

5

fiber size, foci of fiber necrosis, regeneration of muscle fibers, centrally nucleated fibers, and hyaline fibers. There is also an increase in connective tissue and fat. In later stages, fat and connective tissue replace almost all muscle fibers. In BMD, the histopathology is milder with less fiber necrosis but more of fiber splitting, regenerating muscle fibers and gross fiber size variation. Immunohistochemistry with antibodies against different dystrophin domains shows absence or very little labeling in patients with DMD, whereas in BMD there is labeling that may be reduced and has varying intensity. In intermediate forms of DMDIBMD there is a weak labeling in most muscle fibers, congruent with a dystrophin quantity higher than in DMD but smaller than in BMD. Female carriers for DMD present a mosaic pattern on immunohistochemistry (Arahata et al., 1989). 21.3.5. Genetic testing The commercial genetic tests for DMD currently available can detect 65 to 70% of the mutations. For detection of deletions, PCR is used in most laboratories, and can detect 98% of the cases (Chamberlain et aI., 1988); Southern blot is also used. These methods are also used in identifying duplications. However, to detect small mutations by DNA-testing further methods are required. Recently an enhanced method for detection of small mutations, which may increase the sensitivity to 90% was reported (Mendell et al., 2001). In 93 patients with DMD without identifiable gene mutations, a highly sensitive DNA analysis (single-strand conformation polymorphism method), revealed small mutations in 73%. Mutations were found throughout the dystrophin gene. Most mutations were new and earlier not reported.

The authors conclude that the findings, together with safety aspects and cost savings, are in favor for DNA analysis as the initial diagnostic test for suspected dystrophinopathy.

21.4. Management and treatment options 21.4.1. Management Boys with DMD will through their childhood and youth need support from physiotherapists and occupational therapists through their life in order to be assessed and find aids and ways to cope with the situations of daily life. Physiotherapists must instruct the parents in passive exercises through a full range of movements; night splints for the ankles may be helpful (Hall, 1984). Prolongation of walking when this is about to become impossible, may be achieved by the use of calipers (Dubowitz, 1989a). Orthopedic assistance is needed for making orthoses like splints and calipers, as well as for surgery of contractures. As the child becomes wheelchairbound there is rapid progress of contractures, and scoliosis develops. A spinal jacket may reduce scoliosis, but surgery might still become necessary. Some DMD boys have urinary dysfunction because of an upper motor neuron lesion due to severe scoliosis or a complication of spinal fusion surgery (Caress et al., 1996). Whenever surgery is undertaken for various reasons, rapid mobilization and intensive postoperative physiotherapy are essential for the outcome. The school situation and social activities for the DMD patients must be supported so that the patient will experience a good quality of life despite physical limitations and cognitive problems. An electric wheelchair is necessary for the prolongation of independence, although the patient may be totally dependent on others in activities of daily life.

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The respiratory insufficiency that develops with time requires ventilator support during the night. Around the DMD patient there is generally a team of professionals to tackle all the varying needs and symptoms arising along the path as the disease progresses. Before the era of molecular genetics a family could come down with several children with DMD, seeing them pass away one after the other. Genetic counseling can be given to families already having a child with DMD. It is important with early diagnosis of DMD in families since this may prevent the birth of further affected siblings. Carrier females have a 50% chance of transmitting the DMD mutation with each new pregnancy. One third of new DMD cases are caused by a new mutation in the mother, and one third by a new mutation in the affected child (Anderson, 2002), thus only about one third of new cases have a known family history The situation in BMD is varying depending on age of onset and severity, and the different types of support already mentioned may be needed sooner or later. Some BMD patients may need a heart transplant. Patients with XLDC need cardiac surveillance and medical treatment, and affected young males may need a heart transplant.

21.4.2. Steroids No specific therapy for DMD is available. However, since 1974 steroid treatment in DMD has been used (Drachman et al., 1974; Brooke et al., 1987; DeSilva et al., 1987; Fenichel et al., 1991; Angelini et aI., 1994; Dubrovsky et al., 1998). Different steroids and various doses and length of treatment have been used in the hope of obtaining maximal effect with minimal side-effects. The most common side-effects are obesity, osteoporosis and cataract. In a recent double-blind randomized study deflazacort and prednisone treatment were compared (Bonifati et al., 2000). Eighteen DMD patients aged 5.2 to 14.6 years (mean 7.3) were treated with deflazacort (0.9 mg/kg/day) or prednisone (0.75 mg/kg/day) for one year. The difference in dosage for the steroids was based on the fact that deflazacort is less potent, the potency ratio being 1: 1.3 (Markham and Bryson, 1995). Untreated DMD patients with a mean age of 6.9 years were used as controls. Muscle strength was evaluated with the Medical Research Council (MRC) scale in four limb muscles, and

performance tests assessing gait, Gowers' maneuver, rising from a chair and climbing four steps were carried out every third month. Body weight and length and blood pressure were monitored. After one year both steroids proved to be equally effective as to MRC scale and functional tests. In the control group a steady and progressive downhill course had taken place during the same year. The side-effects included weight gain which was significantly higher in the prednisone group. Skeletal bone age was similar in the treated groups. Slight cataract was seen in two boys in the deflazacort group and in one with prednisone treatment. Blood tests such as CK, glucose, etc. did not significantly differ between the groups. Gastrointestinal pain due to steroid treatment did not differ among the treatment groups. No other side-effects were noted during the one-year trial. A long-term effect of steroid treatment was reported in a retrospective study by Shara et aI. (2001). Thirteen of 19 DMD boys aged 8.9 to 18.1 years and treated with 0.9 mg/kd deflazacort daily for 49-70 months (mean 65 mo) were compared to 13 untreated age-matched DMD boys. Muscle strength and functional tests were significantly better in patients on steroids. The vital capacity was significantly improved in the treated group. Cardiologic examinations did not show any difference between the groups. The main side-effects of steroids were short stature (11 of 13), cataracts (6 of 13) and obesity (2 of 13). The exact anti-inflammatory mechanism of steroids in DMD has not been identified. One report shows that prednisone prevents the action of infiltrating dermal dendritic cells (Matasic et al., 1999), which has been reported to occur in dystrophic muscle (Chen et al., 2000). However, this would only partly explain the action of steroids in DMD.

21.4.3. Other medication trials Quite recently the use of the aminoglycoside gentamicin has been tried in DMD. In animal models gentamicin has been shown to interfere with ribosomal recognition of stop codons and can readthrough the stop codons (Howard et al., 2000). Some DMD are caused by nonsense mutations producing premature stop codons, and gentamicin would possibly lead to production of dystrophin. A trial on four patients given gentamicin during two weeks did not

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show any increase in muscle strength or dystrophin levels (Wagner et aI., 2001). More animal studies are recommended, and possibly the development of a less toxic aminoglycoside, before further trials on humans are carried out (Karpati and Lochmuller, 2001). 21.4.4. Gene therapy research for DMD

The goal of gene therapy in DMD would be to rescue muscle tissue by providing a normal dystrophin cDNA to the muscle fibers before the regenerative capacity of muscle fibers totally fails. Much research has utilized the mdx mouse, which lacks dystrophin due to a mutation on exon 23 leading to production of a shortened, unstable dystrophin protein (Karpati et al., 1997). The diaphragm muscles of these animals are severely affected. By making the mdx mouse transgenic through providing copies of mouse or human cDNA to the fertilized egg, the strength of diaphragm in transgenic mdx mice showed no statistical difference to that of normal mice (Hauser and Chamberlain, 1996). Myoblast transfer studies imply injection of myoblasts with normal dystrophin into muscle, where the myoblasts fuse with muscle fibers and produce dystrophin. However, there are problems connected with this method, such as death of the myoblasts shortly after injection, immune rejection, and difficulty in producing enough myoblasts in culture. Gene transfer with recombinant dystrophin genes, i.e. functional dystrophin genes which are reproduced in vitro and delivered into muscle by means of viral vectors, has been a hot issue over the past years. Viral vectors used in this research are retrovirus, adenovirus, herpes simplex virus, and an adenoassociated virus. Currently, the most promising gene delivery vector is the adeno-associated virus which is nonpathogenetic, and shows safety and good properties for gene transfer into muscle (Greelish et aI.,1999).

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