Keywords

Introduction

The dystrophinopathies include a spectrum of muscle diseases caused by mutations in dystrophin gene (DMD) at Xp21 which encodes the protein dystrophin. The mild end of the spectrum includes the phenotypes of asymptomatic increase in serum concentration of creatine kinase (CK) and muscle cramps with myoglobinuria and isolated quadriceps myopathy. Variable phenotypic expression in dystrophinopathies relates mainly to the type of mutation and its effect on the amount of functional dystrophin production. Duchene muscular dystrophy (DMD) is in the most severe end of the spectrum and is typically associated with <5 % of normal levels of dystrophin in skeletal muscle. The diagnosis is made in most patients at approximately 5 years of age when their physical ability lacks behind their peers. In the milder allelic form Becker muscular dystrophy (BMD), dystrophin mutations do not disrupt the open reading frame; a shortened but functional dystrophin protein is produced, enabling most patients to remain ambulatory until the age of 15. Clinical phenotypes of other patients, so-called intermediate or outliers that do not fit the typical DMD or BMD, also exist. In general, manifesting carriers are in the milder end of the spectrum. Some dystrophin mutations cause an isolated cardiac phenotype resulting in DMD-associated dilated cardiomyopathy (DCM) when the heart is primarily affected.

DMD is the most common form of childhood muscular dystrophy with an estimated birth prevalence of about 1 in 3,500 (2.9 per 10,000) live male births [1]. A recent two-tier system of analysis for newborn screening for DMD that included a large cohort in the United States reports a lower incidence rate, 1 in 6,000 [2]. The disease carries the name of the French neurologist, Duchenne, who published his first description of DMD in 1861 with a more comprehensive account in 1868, establishing diagnostic criteria and accurately describing muscle biopsy features. British neurologist Gowers, in 1886, illustrated the characteristic features of calf muscle hypertrophy and described the way in which an affected child rises from the floor to reach the standing posture [3]; this sign was subsequently named after him. A milder form of the disease with later onset was recognized by Becker and Kiener in 1955 [4]. In the second half of the twentieth century, the histopathological characterization became more precise and the associated marked increases in CK were recognized in affected males and in carrier females to a lesser degree.

Advances in molecular genetics led to mapping of the gene responsible for DMD to band p21 of the short arm of the X chromosome [5–8] and then to the cloning of its DNA sequence in 1987 [9]. Subsequently, Kunkel’s group was able to identify the muscle protein dystrophin encoded by the cloned gene [10], localized dystrophin in the sarcolemma of muscle fibers, and demonstrated its almost complete absence in DMD and its decrease in BMD. These advances permit unequivocal diagnosis of DMD and related phenotypes and allow accurate genetic counseling, reliable prenatal testing, and newborn screening.

Etiology and Pathogenesis

All dystrophinopathies are allelic conditions, resulting from different mutations of the dystrophin gene.

Dystrophin

Dystrophin is a scaffolding protein in muscle encoded by the DMD gene. The gene is the largest identified to date in humans, spanning approximately 2.4 megabases on the short arm of the X chromosome (Xp21). The DMD gene contains 79 exons encoding for a 3685 amino acid, 427 kDa protein [9]. Dystrophin has four primary functional domains which together link the actin cytoskeleton to the extracellular matrix to provide stability and strength to muscle fibers (Fig. 56.1). These include (1) the amino-terminal actin-binding domain; (2) the central rod domain comprised of 24 triple helical spectrin-like repeats interspersed by 4 putative hinge domains imparting flexibility [11]; (3) the cysteine-rich domain containing two EF hands [12], a WW domain [13], and ZZ domain [14] important for signaling and binding β-dystroglycan, the link to the extracellular matrix; and (4) the carboxy-terminal domain which binds critical structural and signaling molecules the syntrophins and α-dystrobrevin [15].

Fig. 56.1
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The dystrophin-associated protein complex. The DAP is a large protein network that spans the sarcolemmal membrane comprised of dystrophin and interacting proteins to stabilize muscle fibers during contraction and relaxation. NH 2 amino-terminal domain, COOH carboxy-terminal domain, ABD actin-binding domain, SR spectrin repeats, CYS cysteine-rich domain, F-actin filamentous actin, nNOS neuronal nitric oxide synthase

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The dystrophin gene can generate several tissue-specific isoforms of different molecular weight, each driven by a distinct promoter (Table 56.1) [16–18]. There are four full-length, large molecular weight (427 kDa) dystrophins: M-dystrophin (Dp427m) present in skeletal and smooth muscle [9], C-dystrophin (Dp427c) in the cerebral cortex and hippocampus [19], P-dystrophin (Dp427p) in Purkinje cells [20], and L-dystrophin (Dp427l) in lymphocytes [21]. Alternative promoters drive expression of five nonmuscle truncated isoforms including Dp260 found in the retina [22], Dp140 expressed in the central nervous system and kidney [23], Dp116 in Schwann cells [24], and two ubiquitously expressed forms, Dp71 and Dp40 [25, 26]. Dp71 is the most abundant isoform in the brain [17] and mutations that affect its expression correlate with the severity of mental retardation in DMD and BMD patients [27].

Table 56.1 Dystrophin isoforms
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The Dystrophin-Associated Protein Complex

Dystrophin is part of a large, tightly associated glycoprotein complex containing structural and signaling proteins (Fig. 56.1), referred to as the dystrophin-associated protein (DAP) complex. The DAP is a large protein network that spans the sarcolemmal membrane stabilizing it during contraction and relaxation [28, 29]. This complex system, ­comprised of three subcomplexes, links the intracellular cytoskeletal actin to the basal lamina, through the extracellular matrix (ECM). The ECM is situated between the sarcolemma and the extracellular basal lamina composed mainly of laminin, collagen, fibronectin, and proteoglycans. Several forms of muscular dystrophy are caused by defects in the function and assembly of the DAP complex [28, 30]. As a central molecular scaffold of the DAP, dystrophin is critical to the structural integrity of the sarcolemma. Severing this link from the subsarcolemmal actin cytoskeleton to the ECM results in secondary deficiencies in other components of the DGC [28, 31]. The DAP can be classified into three domains: extracellular including laminin and α-dystroglycan, transmembrane (sarcoglycans and β-dystroglycan), and subsarcolemmal (dystrophin, syntrophins, dystrobrevins).

Skeletal muscle laminin is a heterotrimer made up of β1-, γ1-, and α2-laminin subunits which binds dystroglycan to the surface of muscle cells [32]. Mutations in laminin-α2 (also designated as merosin) have been described in a subset of patients with congenital muscular dystrophy [33]. In addition to dystroglycan, α7β1 integrin has been identified as a receptor for laminin [34]. Dystroglycan is composed of two subunits generated from a common precursor and cleaved to form α-dystroglycan, which binds to laminin-α2, and β-dystroglycan [35], which spans the membrane and binds to the cysteine-rich domain of dystrophin on the subsarcolemmal side [36].

The sarcoglycan group, comprised of four sarcolemmal transmembrane proteins (α-, β-, γ-, δ-sarcoglycans) along with the stabilizing protein sarcospan [37], demonstrates side-association linkage with dystroglycan [38]. Although the exact role of the sarcoglycans in the molecular organization of the DAP is not well defined, it is clear that it plays both structural and signal transduction roles [39]. Deficiency of the sarcoglycan complex proteins is responsible for ­several limb-girdle muscular dystrophies, known as sarcoglycanopathies, and secondary deficiencies of the sarcoglycans in DMD patients likely contribute to the dystrophic ­phenotype [39]. Dystrophin itself resides in the subsarcolemmal domain binding actin through its N-terminal domain and intracellular signaling molecules, the syntrophins [40], dystrobrevins [41], and neuronal nitric oxide synthase (nNOS) [42] via its C-terminal domain. The quantity of dystrobrevin is reduced in both DMD and patients with sarcoglycanopathies [41].

Dystrophin Gene Mutations

Accurate dystrophin mutation analysis is critical not only for diagnostic purposes but also for considering potential treatments due to mutation specificity. Dystrophin gene mutation analysis reveals a predominance of deletions of one or more exons in 60–70 % of DMD/BMD cases primarily clustering in two hotspots of the gene, proximal (exons 2–20) and distal (exons 45–55) [43, 44]. Duplications resulting in in-frame or out-of-frame transcripts account for 6 % of mutations in DMD and for about 10 % in BMD [44]. Point mutations (which can include small deletions or insertions, single base changes, and splice-site mutations) account for ∼25–35 % of mutations in DMD but are less common in BMD, comprising about 10–20 % of cases [44–46]. Nonsense mutations occur more commonly in DMD, in approximately 20–25 % of cases as compared to <5 % in BMD [44, 46]. Splice-site mutations and small insertions or deletions account for a considerable proportion of sequence changes found in both DMD and BMD while missense mutations are rare. DMD-associated dilated cardiomyopathy (DCM) is caused by mutations in DMD that affect the muscle promoter (PM) and the first exon (E1), resulting in no dystrophin transcripts being produced in cardiac muscle; however, two alternative promoters that are normally only active in the brain (PB) and Purkinje cells (PP) are active in the skeletal muscle, resulting in dystrophin expression sufficient to prevent manifestation of skeletal muscle symptoms [47–49]. DMD-associated DCM may also be caused by alteration of epitopes in a region of the protein of particular functional importance to cardiac muscle [50] or possibly by mutations in hypothetic cardiac-specific exons [51].

Germline Mosaicism

Dystrophinopathies are familial diseases; however, sporadic cases do occur resulting from a spontaneous mutation in a single germ cell of the mother or perhaps in the proband’s dystrophin gene early in postzygotic embryo development. If the mother is not a somatic carrier but has more than one affected offspring, that may be related to the phenomenon of germline mosaicism [52, 53]; the latter accounts for about 20 % of new DMD mutations [54, 55]. This phenomenon may be due to a mutation at a mitotic stage of germ cell lineage development. If this type of mutation occurs in a female, there is a substantial risk for more than one affected male offspring. If the germline mutation occurs in a male, his female offspring are at significant risk for becoming carriers [53]. Because of the possibility of germline mosaicism, if the mother of an affected male is not a carrier by deletion analysis of her lymphocyte DNA, her risk of having another affected son could be at least 7–10 % [56].

Dystrophinopathies in Females

During early embryonic development, random inactivation of one of the two X chromosomes occurs (lyonization), leaving active 50 % of the maternally derived chromosomes and 50 % of the paternally derived ones [57]. Thus, if the maternal X chromosome carries a mutated dystrophin gene, 50 % of the remaining nuclei have an active normal dystrophin gene and thus will produce enough dystrophin to prevent muscle necrosis [58]. In the case of nonrandom X chromosome inactivation, less than 50 % of the nuclei may have the normal dystrophin gene in an active state, thus resulting in dystrophin deficiency and clinical manifestations in females [59]. This happens most commonly in cases of twin pregnancies [59]. Cases of X-autosomal translocations with breakpoints at Xp21 resulting in preferential inactivation of the normal chromosome have also been described [5, 8]. In Turner syndrome (karyotype XO) or Turner mosaic (XO/XX or XO/XX/XXX) syndrome, if the X chromosome carries a mutated dystrophin gene, the female child will be clinically affected also.

Pathogenesis

More than a decade prior to the cloning of dystrophin, ultrastructural observations were made in DMD muscle biopsies which alluded to the pathogenesis of the disease. In 1975, Mokri and Engel noted focal plasma membrane defects in nonnecrotic fibers in DMD muscle [60]. These regions, referred to as delta lesions, were accompanied by nearby cytoplasmic defects where myofibrils were usually contracted. Histopathological findings and experiments in the mdx mouse model of DMD support the conclusion that reduced or absent dystrophin results in a mechanically weakened plasma membrane [61, 62], which is prone to focal tears during contractile activity [63]. This allows a massive influx of extracellular calcium, which activates proteolytic enzymes and leads to gradual necrosis of muscle fibers explaining why dystrophinopathies are progressive diseases [64]. A second aspect of DMD pathology is functional ischemia which was proposed as a pathogenic mechanism of muscle fiber damage in DMD over 30 years ago based on experimental findings reproducing the histopathological pattern of muscle necrosis and regeneration [65]. Recent studies reinforce the potential role of ischemia through molecular-based findings demonstrating deficiency of nitric oxide synthase (NOS) in DMD muscle as a contributory factor in muscle damage [66]. As a result, therapeutic strategies helping to circumvent ischemia by modulation of the NOS pathway are emerging (see section “Emerging Therapies”).

Clinical Presentation

There is a great heterogeneity in the clinical presentation and course of the various dystrophinopathies, creating a spectrum ranging from very severe to very mild presentations.

Duchenne Muscular Dystrophy

In children with DMD, although there is histologic and ­laboratory evidence (elevated serum CK ≥ 2,000 [2]) of myopathy at birth, clinical manifestations are usually absent at this age. During the first 2 years of life, some affected boys exhibit mildly delayed gross motor development presenting primarily as delayed walking. The mean age of walking is approximately 18 months (range 12–24 months). Weakness is usually noted between 2 and 3 years of age; in some cases, however, it may be delayed and become apparent after the age of 3 years (Table 56.2). The first symptoms are usually difficulty with running, jumping, and going up the stairs, related to weakness that selectively affects proximal limb muscles before distal and the lower extremities before the upper ones. Between 3 and 6 years of age, a broad-based waddling gait, exaggerated lumbar lordosis, and calf enlargement are usually observed. Early on, toddlers may complain of leg pains. In arising from a supine position on the floor, affected boys turn their face to the floor, spread their legs, and use their hands to climb up their thighs to an upright position (Gowers’ sign) (Fig. 56.2). Neck flexor weakness often goes unnoticed but it does occur at all stages of the disease; when lying on his back, a child with DMD is unable to lift his head against gravity [67]. This sign is helpful in distinguishing boys with DMD from patients with milder phenotypes, such as “outliers” and BMD.

Table 56.2 Genetic, clinical, and laboratory features of the dystrophinopathies
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Fig. 56.2
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A boy with Duchenne muscular dystrophy demonstrating Gowers’ sign. To rise from the floor, he is faced to the floor, his legs are spread, and his buttock is raised; subsequently he will use his hands to climb up his thighs (Courtesy of Dr. John Kissel, The Ohio State University, Columbus, OH)

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Physical examination shows firm enlargement of the calf muscles and, in some instances, of the quadriceps, gluteal, ­deltoid, and rarely masseter muscles. Increased muscle bulk, early in the course of the disease, is the result of true hypertrophy, however later on is related to replacement of the muscle fibers by fat and connective tissue (pseudohypertrophy) [68]. The ankle plantar flexors and invertors remain very strong throughout the course of the disease, while the tibialis anterior muscles become weak gradually, resulting in heel cord contractures and toe walking; eventually, this imbalance in muscle strength will lead to bilateral equinovarus foot deformities.

Between 3 and 6 years of age, there may be some improvement; this is related to normal motor development of the child which outpaces disease evolution, to be ­followed gradually by relentless deterioration. Between 6 and 11 years, muscle strength decreases linearly and the ability to climb stairs, rise from a supine position, climb stairs with rails, and walk a short distance declines rapidly in the mentioned order [54, 68]. As the disease progresses, muscle stretch reflexes diminish or cannot be elicited; before the age of 10 years, the triceps, biceps, and knee reflexes become hard to elicit in approximately 50 % of patients. The brachioradialis stretch reflex remains active longer with the ankle reflex being elicited in one-third of patients even during the last phase of the disease [69]. Cranial and sphincter muscles are spared [70].

The development of joint contractures is another almost constant clinical feature of DMD. By age 6 years, the majority of DMD patients have contractures of the heel cords, iliotibial bands, and hip joints, causing toe walking and limitation of hip flexion. By age 8 years, knee, wrist extensor, and elbow contractures appear and correlate with decreasing ambulation [68]. Shoulder contractures occur only late in the disease course. At around age 10 years in most patients, the weakness leads to dependency on long leg braces for ambulation [68] and wheelchair confinement by approximately the age of 12 years [71]. The use of steroids prolongs ambulation 1 to 2 years. Scoliosis rarely occurs prior to age 11 while they are still ambulatory; severe curvatures usually evolve after wheelchair confinement requiring medical intervention [72]. Increasing scoliosis and gradual deterioration of pulmonary function related to the weakening of respiratory muscles are leading cause of respiratory failure. Patient’s age at respiratory failure correlates well with the degree of thoracic kyphoscoliosis [73].

Historically the patients, usually around age 20, succumbed to restrictive lung disease or complications but also died from cardiac failure secondary to progressive cardiomyopathy [74], but now improved care, including antibiotics, vaccines, and other ancillary methods, protects the cardiorespiratory system and prolongs life.

Involvement of Other Organ Systems

Heart, brain, and smooth muscle are also involved in the ­disease process, resulting from the lack of dystrophin ­expression in these organs.

Cardiac involvement is inevitable in DMD and the incidence of cardiomyopathy increases steadily in the teenage years. Approximately one-third of patients are affected by age 14 years, one-half by age 18 years, and all after age 18 years [75]. The majority of patients, however, remain free of cardiovascular symptomatology until late in the disease, probably due to their inability to exercise, which may mask the cardiac symptoms [54]. In the late stages of the disease, congestive heart failure and arrhythmias may develop, especially during infections. In very rare cases, congestive heart failure dominates the picture and can be the immediate cause of death without marked compromise of respiratory function [68].

At the time when boys with DMD are wheelchair dependent, the dystrophin-deficient myocardium may or may not demonstrate measurable changes by echocardiography [76]. In contrast, electrocardiographic (ECG) abnormalities can be detected from a young age (<6 years of age), identified in about 76 % of patients. The most common ECG abnormality noted in very young boys is related to left ventricular pathology, manifesting most commonly by a Q wave >98th percentile in lead III or V6 [77]. Prominent Q waves in leads II, aVF, and V5 were also reported [78]. The characteristic changes in older boys include short PR interval, right ventricular hypertrophy, and deep Q waves in leads I, aVL, and V5 [71, 72]. Intra-atrial conduction disturbances, sinus tachycardia, or other sinus arrhythmias are more frequent than atrioventricular conduction defects and infranodal/ventricular abnormalities. ECG changes are similar in patients with DMD regardless of presence of dilated cardiomyopathy (diagnosed by an ejection fraction <55 %) suggesting that ECG may not be predictive of disease course [78].

Autopsy studies of the heart in patients with DMD show the initial site of myocardial fibrosis and fatty infiltration to be in the posterobasal epicardium, progressing to involve the epicardial half of the left ventricular free wall, then occasionally involving septal segments as the fibrosis becomes more severe and more transmural [76, 79]. The right ventricle and the atria seldom are involved. Fibrosis and fatty infiltration can also involve the conduction system, the sinoatrial and the atrioventricular nodes [80, 81]. As in autopsy studies, ­cardiovascular magnetic resonance studies find that scar ­predominantly involves left ventricular segments and that septal segments are involved less frequently and only in patients with significant left ventricular free wall involvement [82]. Ultimately the fibrosis leads to dysfunction and dilation, to a severe generalized cardiomyopathy. In some instances, the left ventricle dilatation cannot occur if the wall is severely fibrotic, creating a restrictive myopathy. With dilation, patients can develop mitral regurgitation and, occasionally, aortic regurgitation. With left ventricular failure, there can be secondary pulmonary hypertension and right ventricular failure associated with pulmonary and tricuspid regurgitation [83].

Most patients with DMD exhibit nonprogressive impairment of intellectual function, initially described as a general leftward shift in the spectrum of IQ scores [84], reduced approximately one standard deviation from the normal population. In some cases, an occasional child with DMD may have average or above-average intelligence.

Although the neuropsychological profile of DMD has not yet been fully characterized, studies show significantly lower performances in verbal IQ, verbal short-term memory, and phonological abilities, as well as in praxis and executive functioning domains [85]. These deficits in executive function are often confused with attention deficit/hyperactivity disorder warranting appropriate neuropsychological testing for correct diagnosis. A neuropathologic abnormality underlying the cognitive deficit in DMD has not been found [68]. Loss of Dp71, the major DMD gene product in brain, is thought to contribute to the severity of cognitive impairment [27]. Studies in animal models suggest a role for Dp71 in excitatory synapse organization and function [86].

Degeneration of gastrointestinal tract smooth muscle resulting from dystrophin deficiency may lead to an important and even life-threatening complication, intestinal hypomotility, also known as intestinal pseudo-obstruction. It may present with acute gastric dilatation, vomiting, abdominal pain, and distension [68, 87]. Degeneration of the outer, longitudinal, smooth muscle wall of the stomach has been documented pathologically.

Osteoporosis in boys with DMD begins to develop early, while they are still ambulating; it is more severe in the lower extremities and may lead to frequent fractures that, sometimes in older children, hasten the loss of ambulation [88]. Corticosteroids further increase the risk of vertebral compression fractures.

Becker Muscular Dystrophy

BMD has been estimated to occur approximately one-tenth as frequently as DMD with an incidence of 1 individual per 30,000 male births (Table 56.2). Compared to fairly stereotypical clinical features of DMD, BMD comprises a more heterogeneous group, which can vary from mildly symptomatic forms to more significant muscular and cardiac involvement. In BMD, the age of onset of symptoms is later than DMD, usually between the ages of 5 and 15 years, sometimes even in the third or fourth decade or later [89]. Most patients with BMD remain ambulatory until 15 years of age and older, although this is an arbitrary cutoff, since these disorders represent a continuous spectrum related to the quantity and quality of dystrophin. In most cases dystrophin is reduced in amount and size [90, 91]. The pattern of muscle wasting is similar to DMD. Pelvic girdle and thigh muscles are involved first and calf muscle pseudohypertrophy occurs early in most patients. Tibialis anterior and peroneal muscle groups are less affected. Shoulder girdle weakness develops later after the onset of proximal lower extremity weakness. One exception is the relative preservation of neck flexor muscle strength, a helpful distinguishing feature of the BMD from DMD; neck flexors become weak later in the disease. Contractures are less likely to develop, and scoliosis is also less common, but becomes evident after the patient is confined to a wheelchair. Muscle pain can be very prominent in some patients with BMD and myoglobinuria occurs infrequently [92]. Serum CK levels can be very high, falling into the same range as the patients with DMD (usually >20 to 75 times normal).

Mental retardation is not as common or severe as in DMD, and gastrointestinal symptoms are essentially absent. Cardiac involvement is that of a dilated cardiomyopathy, sometimes starting with right ventricular dilation and progressing to a generalized dilated cardiomyopathy [93, 94]. Recent cardiac magnetic resonance imaging studies in BMD patients show many of the same findings seen in DMD, including fibrosis, and are more sensitive in detecting abnormalities of ejection fraction than echocardiogram [95]. The ECG findings in patients with BMD are also similar to those in patients with DMD with prominent Q waves in I, aVL, and V6, or in II, III, and aVF; tall R waves in V1; and increased QT dispersion [96]. Rarely, cardiac symptoms can be severe in patients with only mild skeletal muscle weakness [97–102] or can precede muscle weakness by several years [103]. The mean age at cardiomyopathy diagnosis is around 14 years, similar to that in DMD [104]. Despite the milder skeletal muscle involvement, heart failure from DCM is a common cause of morbidity and the most common cause of death [105]. Patients usually survive beyond the age of 30 years [68], with death from dilated cardiomyopathy/cor pulmonale or respiratory insufficiency occurring between 30 and 60 years [105].

Earlier studies have reported the presence of cardiac involvement with BMD is associated with deletions in two regions of the DMD (5′ end and exons 47 through 49) [93]. An extensive analysis of genotype–phenotype correlation in BMD patients shows that early-onset cardiomyopathy is associated with mutations in the amino-terminal domain; in contrast deletions removing portions of the rod domain along with the hinge 3 domain have a later onset, likely due to preservation of the spectrin-repeat structure of the protein [51].

Late-onset dystrophinopathy could in rare instances present with a myopathy involving predominantly, but not exclusively, the quadriceps [106–108]. Some of these patients may have cardiac involvement. In earlier reports of four patients, dystrophin deficiency, proved by immunoblot analysis, was demonstrated to be the cause of the myopathy which was considered “atypical form” of BMD [106]. Several other cases of isolated quadriceps myopathy have been reported since then and proven to be due to mutations in the dystrophin gene [107–109], thus demonstrating that quadriceps myopathy can sometimes be the only manifestation of dystrophin deficiency.

X-linked exercise-induced myalgias and cramps without skeletal muscle weakness, starting in childhood, especially in a family with cardiomyopathy, can be caused by mutations in the dystrophin gene [110, 111]. However, a longer follow-up period is necessary to definitely rule out late-onset skeletal muscle weakness, which could classify these cases as BMD [103, 110]. Two molecular regions on the dystrophin gene have been linked to the phenotype of cramps and myalgias: exons 10–44 [109, 110] and exons 45–52 [103, 111]. Some of the patients in these studies also had fixed weakness [109].

Dystrophin Gene-Associated Dilated Cardiomyopathy

X-linked DMD-associated DCM is a progressive and fatal type of heart disease that presents in the second or third decade of life with congestive heart failure in patients with minimal skeletal muscle symptoms [47, 112–114]. It should be noted that four separate genetic disorders with cardiac and skeletal myopathies are X-linked. DMD and BMD already have been discussed. Those with no or minimal abnormalities in skeletal muscle dystrophin but showing abnormal dystrophin in heart muscle are classified here as DMD-associated DCM. The other X-linked cardiomyopathies include Emery-Dreifuss muscular dystrophy, resulting from abnormalities in LINK components at nuclear envelope [115, 116], and Danon disease [117], also known as X-linked vacuolar cardiomyopathy and myopathy, which is caused by a mutation in the gene encoding lysosome-associated membrane protein-2 (LAMP2). Another disorder of this group is Barth syndrome [118] a mitochondrial disease caused by a mutation of the tafazzin gene associated with decreased amounts and altered structure of cardiolipin, the main phospholipid of the inner mitochondrial membrane.

Boys with DMD-associated DCM complain of exercise intolerance, muscle fatigue, pain, and cramping but do not manifest the weakness seen in patients with BMD and DMD [119]. Calf hypertrophy and elevated serum CK levels can be present [94]. Symptoms of congestive cardiomyopathy usually develop in late teen years to early adulthood that usually progresses to death within 2 years of the onset of the myopathy diagnosis. Arrhythmias and atrioventricular block can occur with much greater frequency than that seen in patients with either DMD or BMD. Female carriers develop mild dilated cardiomyopathy later in life with slow progression and often fatal outcome [47, 105].

Different mutations in the dystrophin gene cause selective absence of dystrophin in heart muscle [47, 54]. A review of families with DMD-associated DCM points out that mutations involving the 5′ end of the gene result in more severe cardiomyopathy than mutations in the spectrin-like region (centered around exons 48–49) [120]. With mutations involving the 5′ end of the dystrophin gene, the exclusive cardiac involvement seems to be related to a difference in RNA splicing regulation between heart and skeletal muscle. The skeletal muscle maintains dystrophin production by using exon skipping or alternative splicing, while the heart muscle is apparently unable to employ such mechanisms. Studies demonstrated that in individuals with the most severe cardiac phenotype the cardiac muscle is usually unable to produce functional dystrophin in the heart, while in skeletal muscle reduced levels of virtually normal dystrophin transcript and protein are present [121, 122]. Classification of DMD-associated DCM as a separate entity in dystrophinopathy spectrum is somewhat controversial. DMD-associated DCM may be the presenting finding in individuals with BMD who have little or no clinical evidence of skeletal muscle disease. Some investigators classify such individuals as having subclinical or benign BMD, whereas others may classify them having DCM with increased serum CK concentration [123].

Manifesting DMD/BMD Carrier Females

Carriers are usually free of symptoms but may have mildly increased serum CK and usually mild calf hypertrophy. In approximately 8 % of the cases they can, however, present with mild myopathy of the limb-girdle type or even typical DMD/BMD [124–128]. Cardiac involvement is usually subclinical, although few investigators have evaluated DNA-proven DMD and BMD female carriers. Earlier studies demonstrated that 8 % of DMD carriers have dilated cardiomyopathy versus none in BMD carriers. Only 38 % of the studied carrier population had a completely normal heart investigation. The remaining had subclinical EKG or echocardiographic abnormalities [129]. Severe cardiac symptoms can occur in some carriers [130].

A more recent study shows DMD carriers (mothers) can have significant left ventricular systolic dysfunction, which is unmasked by exercise, [131] implying the need for evaluation of proven female carriers, even if they are asymptomatic.

Differential Diagnosis

The process of making an accurate diagnosis in suspected DMD/BMD patients depends on the mode of presentation, in particular the pattern of muscle involvement, and additional clinical features, the serum CK level, and any informative family history. If there is clear evidence for autosomal dominant transmission, a dystrophinopathy can easily be excluded. If not, a diagnostic workup may include a muscle biopsy. Immunohistochemistry with antibodies against dystrophin epitopes, α-, β-, γ-, and δ-sarcoglycans, α- and β-dystroglycans, and merosin (laminin-alpha 2) may offer a specific biopsy diagnosis. Western blot analysis is useful for quantitation of dystrophin, calpain, or other proteins. However, DNA mutation analysis should ultimately be done to confirm the results from these biochemical studies.

Salient features of the following disorders that can be considered in the differential diagnosis of the DMD and BMB are discussed below.

Limb-girdle muscular dystrophy (LGMD) encompasses a heterogeneous group of muscle disorders characterized by a predominantly proximal distribution of weakness with the age of onset of symptoms varying from early childhood to late adulthood. Typically, the onset is not congenital. Pathogenic genes reported so far include those that encode integral components of DGC as well as other structural proteins, and even enzymes such as calpain 3. In certain types of LGMD there may be clinical clues, but there is substantial overlap in different forms. Recessive forms are far more common than autosomal dominant forms that have more heterogeneous clinical presentation with some families showing syndromic features.

LGMD2A or calpain 3 deficiencies, the most common recessive form, represent about 10 % of LGMD population and can present within the first decade with very high serum CK levels with a pelvifemoral pattern of weakness. Early contractures and absence of cardiac involvement are helpful clinical features. Presentation of calpainopathy with high serum CK can also occur, usually observed in children or young individuals, in which symptomatic individuals have only high serum CK concentrations. Clinical findings include the tendency to walk on tiptoes, difficulty in running, scapular winging, waddling gait, and slight hyperlordosis.

Other well-characterized LGMD subtypes are sarcoglycanopathies (α-sarcoglycanopathy, LGMD2D; β-sarcoglycanopathy, LGMD2E; γ-sarcoglycanopathy, LGMD2C; δ-sarcoglycanopathy, LGMD2F). Clinical features range from early childhood onset with severe progression similar to DMD to later onset with milder progression as seen in BMD (see Table 56.2). Calf hypertrophy is commonly observed but scapular winging is more prominent than DMD/BMD. Heart involvement is variable, but typically less severe than in the dystrophinopathies. Overall, about 30 % of individuals have evidence of cardiomyopathy by ECG and echocardiogram; cardiomyopathy is common in β-, γ-, and δ-sarcoglycanopathy, but rare in α-sarcoglycanopathy [132, 133].

Most individuals with severe, childhood-onset LGMD are suspected to have sarcoglycanopathy [134]. Thus, a boy with a clinical presentation and progression similar to DMD but with normal dystrophin immunostaining in muscle is likely to have a primary sarcoglycanopathy. In contrast, only about 10 % of individuals with LGMD with milder disease (onset in adolescence or adulthood) have a sarcoglycanopathy.

LGMD2I is an important type of LGMD associated with abnormal α-dystroglycan labeling on the muscle biopsy and caused by mutations in genes encoding proteins involved in the glycosylation of dystroglycan such as FKRP, the gene encoding fukutin-related protein [135]. The phenotype ranges from severe to mild with no clinically apparent skeletal muscle involvement [136–140]. Cardiomyopathy without skeletal muscle involvement has been reported. In severe cases loss of ambulation occurs in the beginning of the second decade. The milder end of the spectrum more closely resembles BMD, with ambulation continuing into the third decade. Cardiac involvement occurs in 10–55 % of affected individuals.

Emery-Dreifuss muscular dystrophy (EDMD) in early childhood typically presents with prominent contractures involving ankles, elbows, and spine together with a slowly progressive muscle weakness and wasting initially in a humeroperoneal distribution and later extending to the scapular and pelvic girdle muscles. In some patients contractures may be less prominent and muscle weakness is more proximal with a slowly progressive course. The cardiac involvement is an important part of the clinical feature that may include palpitations, presyncope and syncope, poor exercise tolerance, and congestive heart failure. A pure cardiac presentation is seen in some families. The X-linked form is caused by mutations in EMD, the gene encoding emerin; the dominant/recessive forms are caused by mutations in LMNA, the gene encoding lamin A/C.

If symptoms of limb-girdle dystrophy are found in a girl, one should always consider the rare possibility of DMD or BMD occurring in a female with an abnormal karyotype (e.g., 45 XO) or nonrandom X chromosome inactivation.

Acid maltase deficiency (AMD) and spinal muscular atrophy (SMA) type III may be considered in the differential diagnosis with proximal muscle weakness and sometimes calf muscle hypertrophy; however, the serum CK, electrodiagnostic studies, and muscle biopsy features (i.e., glycogen storage in AMD, neurogenic changes in SMA) will easily permit the distinction from these disorders [54].

In rare instances, dystrophinopathies may present similarly to congenital muscular dystrophies [3, 141] with neonatal weakness, hypotonia, and subsequent developmental delay. The differential diagnosis can be made clinically by the modestly increased CK level and the frequent facial and eye muscle involvement and contractures in congenital muscular dystrophies. Muscle biopsy immunostaining for dystrophin may also be helpful in differentiating the two entities.

Dermatomyositis before the onset of the rash in rare instances can be mistaken for a dystrophinopathy, but the acute onset of symptoms permits easy distinction from DMD or BMD.

An unexplained persistent elevation of liver enzymes should always alert the clinician to the possibility of an underlying muscular dystrophy including females, who could prove to be symptomatic females, asymptomatic carriers of DMD/BMD, asymptomatic males with DMD/BMD, or patients with other muscular dystrophies [142]. In these instances, measurement of serum CK, which is a more specific muscle enzyme, should always be made to prove the myogenic origin of the elevated liver enzymes. Finally, a dystrophinopathy should be considered in all cases of dilated cardiomyopathy.

Laboratory Features and Diagnosis

The new era of advanced molecular diagnostic tools provides an accurate and prompt diagnosis of DMD/BMD, allowing initiation of appropriate measures, continuing support and education, and avoiding a potentially pervasive diagnostic process. Suspicion of clinical diagnosis for DMD or BMD should be considered irrespective of family history on the basis of the clinical presentation and level of serum CK. Reduced motor skills, particularly the inability of run and jump in a boy between ages 3 and 5 years, bring the possibility of DMD to attention. Older boys with later age of onset for similar pattern of weakness involving pelvic girdle and thigh muscles followed by proximal upper extremity weakness are suspect for BMD diagnosis. In both situations, testing for serum CK is indicated. The next step in the diagnostic strategy should be confirming the clinical diagnosis of DMD/BMD using available new tools for identifying precise genetic defect. Extensive mutation analysis is a necessity, not to satisfy academic interest but because evolving treatment paradigms depend on the full characterization of the deletion/duplication endpoints and the identification and position of point mutations.

Serum Muscle Enzymes

CK is the most important serum enzyme in the diagnosis of DMD. Striking CK elevation may be present even in the first years of life, preceding clinical manifestations of obvious motor impairment. Before the age of 5 years, the serum CK levels are usually 50–100 times of normal in DMD and can be markedly increased to 20–100 times of normal in BMD [68, 143] and, therefore, cannot be used as a way of differentiating between the two types of dystrophy. The concentration of CK, however, tends to decline with age, at a rate of about 20 % per year. In about 70 % of carriers, CK levels are elevated, but decrease with age.

Serum aminotransferases, AST and ALT, both produced by muscle as well as liver cells are also usually elevated in DMD and BMD. Their increased levels are thought to be related to leakage through muscle membranes [142]. Therefore, diagnosis of DMD should be considered before liver biopsy in a male child particularly with unintended discovery of increased transaminases.

Electromyography

Patients with classic features of DMD or BMD do not need electrodiagnostic studies for diagnostic purposes, but in sporadic cases of BMD or carrier females with modest serum CK elevation (less than 1,000 IU/L) and proximal muscle weakness, electromyography may have to be considered to exclude a neuropathic process (e.g., SMA) and before undergoing genetic testing for confirming the diagnosis. Nerve conduction studies including compound muscle action potentials (CMAPs) are normal in the early phases of the disease. Needle EMG in DMD/BMD shows myopathic changes, usually short-duration, low-amplitude polyphasic early-recruited motor unit action potentials (MUAPs), particularly in proximal muscles. It may also show increased insertional activity with fibrillation potentials. With disease progression, the CMAPs decrease in amplitude, the insertional activity diminishes, MUAPs become very small with decreased recruitment, and the fibrillation potentials disappear. At the advanced stages of DMD/BMD, the muscle may become electrically silent.

Molecular Genetic Testing

Until recently, multiplex PCR and Southern blotting were utilized for dystrophin gene deletion/duplication analysis. New tools for mutation detection have been introduced including multiplex ligation-dependent probe amplification (MLPA) and multiplex amplifiable probe hybridization (MAPH) providing relatively rapid and inexpensive exon screening [144]. When MLPA or MAPH are negative, DNA sequencing is required to detect subexonic rearrangements or point mutations [43] (see Dystrophin Gene Mutations).

Muscle Biopsy

Muscle biopsies derived from DMD/BMD patients are processed for examination of histopathological features, immunohistochemistry for detection of dystrophin in muscle membrane, and Western blot analysis for quantitation of dystrophin protein.

Histopathological Findings

Overall the muscle biopsy features of DMD include fiber size variability with atrophy and hypertrophy, an alteration in fiber type resulting in type I fiber predominance, muscle fiber degeneration, regeneration, isolated “opaque” hypertrophic fibers, and significant replacement of muscle by fat and connective tissue. The degenerating necrotic fibers are recognized on H&E and trichrome staining by their lighter-stained glassy or homogenous cytoplasm (Fig. 56.3a). Small groups of basophilic regenerating and necrotic fibers are an important feature of DMD biopsies. Hypercontracted large opaque and darkly stained fibers are seen commonly. Their origin is unclear but thought to be due to segmental hypercontraction resulting from plasma membrane defects that allow the influx of calcium-rich extracellular fluid [145]. Fiber splitting and central nuclei are less often, present in 2–4 % of the fibers compared to other dystrophies.

Fig. 56.3
figure 00563figure 00563

H&E and immunofluorescence staining of frozen sections of skeletal muscle biopsies with anti-dystrophin antibodies. (a–d) From a 5-year-old boy who has DMD with a duplication of exons 55 through 63 in the dystrophin gene. H&E stained section (a) shows a small group of basophilic regenerating muscle fibers (between arrowheads) and necrotic fibers (arrows) among fibers with increased variability in size; asterisk marks a hypercontracted fiber. Membrane staining for dystrophin using specific antibodies to amino-terminal Dys3 (b), rod domain Dys1 (c), and carboxy-terminal Dys2 (d) is absent with the exception of small clusters of revertant fibers. H&E stained section (e) from a 5-year-old boy who has BMD with a duplication of exons 19 through 29 in the dystrophin gene, presented with history of exercise-induced myalgias and CK elevation at 2,000–11,000 IU/L range and no muscle weakness on examination. A small focal area of inflammation is present (arrows). Membrane staining for Dys3 is severely reduced (f), while membrane staining for Dys1 (g) and Dys2 (h) is present, compatible with an in-frame duplication mutation

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Inflammatory cells are seen in the perimysium, endomysium, and perivascular spaces. They consist mostly of T lymphocytes and macrophages. Most T cells are CD8+ and occasional nonnecrotic muscle fibers are focally surrounded and invaded by CD8+ cells [68]. A striking increase in the endomysial and perimysial fibrosis occurs with disease progression. The differences in the microscopic appearance of muscle between DMD and BMD correlate well with the severity of the disease, with fewer necrotic, hypercontracted, and regenerating fibers seen in milder phenotypes and the frequency of hypertrophic fibers and internal nuclei increase with age (Fig. 56.4a).

Fig. 56.4
figure 00564

H&E stained section (a) from a 12-year-old boy with BMD who has exons 10 through 44 and promoters Dp260 and Dp140 in-frame deletion. He is ambulatory and has CK levels over 14,000 IU/L. Marked variability in muscle fiber size (<10 to over 150 μm in diameter), an increase of internal nuclei, and rare necrotic (arrow) or regenerating fibers are seen. Immunofluorescence staining for Dys2 shows normal membrane staining (b); membrane staining for Dys1 and Dys3 is absent (not shown). Membrane staining for utrophin is present suggesting compensatory upregulation (c). Western blot analysis shows a truncated protein reduced in size with Dys2 antibody; dystrophin band is absent with Dys1 antibody (d, lane 2). Lane 3 shows severely reduced dystrophin with Dys1 and Dys2 from a DMD muscle

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Dystrophin Immunostaining

In muscle biopsies derived from DMD patients, there is no detectable staining of the sarcolemma using commercially available anti-dystrophin antibodies against amino-terminal (dys3), carboxy-terminal (dys2), and rod domains (dys1) of the protein. However, in about greater than 50 % of DMD patients, antibodies against different epitopes show membrane staining for dystrophin in about 1 % of fibers in small clusters, which are called “revertant” fibers [146] (Fig. 56.3b–d). They arise from a somatic mutation at a second site of the gene that corrects the original frame shifting mutation and restores the reading frame [147, 148]. In BMD patients, either normal or partial staining of the sarcolemma is observed (Figs. 56.3f–h and 56.4b). Immunohistochemistry for dystrophin is useful in identifying sporadic cases of symptomatic or asymptomatic female DMD carriers with high serum CK levels in families ­without a male proband, or in families with no detectable deletion/duplication results. Thus, symptomatic and asymptomatic DMD carriers with elevated CK values may exhibit a characteristic mosaic pattern of dystrophin immunostaining.

Immunostaining of muscles, from DMD patients or the mdx mouse model of the disease, shows that loss of dystrophin leads to a selective reduction or absence in the staining of the DAPs such as α-, β-, γ-, and δ-sarcoglycans and α- and β-dystroglycans [149]. However, another dystrophin-related protein, utrophin, which has homology to dystrophin shows diffuse membrane staining outside of neuromuscular junctional folds resulting from a compensatory upregulation in dystrophin-deficient muscle [150] (Fig. 56.4c).

Western Blot Analysis

Western blot for dystrophin quantitation accurately predicts the severity of the muscular dystrophy phenotype (Table 56.2), especially in cases with no family history (Fig. 56.4). Because of the exceptions to the reading frame rule, the type of the deletion (in-frame versus out-of-frame) is not always a reliable predictor of the severity of the disease. The Western blot assesses not only the amount of dystrophin but also the molecular size of the dystrophin molecule, which is often decreased (80 %) (Fig. 56.4d) and rarely increased (5 %) in BMD patients (in deletion and duplication cases, respectively). Antibodies against carboxy and rod domains should be utilized in the Western blot assay in order to avoid ­false-negative results. Given the qualitative nature of dystrophin immunostaining and the possibility of normal appearance in patients with BMD, Western blot has become the gold standard for dystrophin analysis.

Algorithm for Diagnosis of DMD/BMD

Familial or Sporadic Cases with Clear Phenotype

If the diagnosis of DMD/BMD has been made clinically in other family members but has not been confirmed previously by molecular diagnosis, the first step is DNA testing for detection of a dystrophin gene deletion/duplication mutation, by MLPA or MAPH (Fig. 56.5). If testing for deletion/duplication is negative, the next step is DNA sequencing to detect subexonic rearrangements or point mutations [43]. The mutation detected in the dystrophin gene may then be used as a marker for testing other at-risk family members, for carrier detection, and also for prenatal diagnosis, by means of amniocentesis or chorionic villus biopsy.

Fig. 56.5
figure 00565

Algorithm to guide diagnostic workup in suspected DMD/BMD patients for confirmation of dystrophinopathy diagnosis. For patients diagnosed by muscle biopsy, dystrophin genetic testing is also necessary. For patients diagnosed by genetic testing, muscle biopsy is not necessary, and if at all possible, defer muscle biopsy until patient ­participates in a later clinical trial. DMD Duchenne muscular dystrophy, BMD Becker muscular dystrophy, CK creatine kinase (*, ** see Table 56.2 for musculoskeletal features; + see Table 56.2 for serum CK levels)

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In sporadic cases (i.e., family history negative for DMD/BMD), the same diagnostic approach, outlined for familial cases, is recommended if the clinical presentation is clear and highly suggestive of DMD or BMD.

Sporadic Cases with Unclear Phenotype

In sporadic cases with unclear phenotype (outliers), if a deletion or duplication is found by MLPA or MAPH, its reading frame status will allow in most instances prediction of the phenotype (DMD or BMD). Because of the rare occurrence of exceptions to the “in-frame/out-of-frame” rule, some clinicians justify the option of a muscle biopsy for Western blot analysis to predict the severity of the disease, although this information has no clinically useful application since these cases represent the spectrum of dystrophinopathy.

If DNA analysis fails to detect a dystrophin deletion/duplication, a muscle biopsy for dystrophin assay will need to be considered, particularly in (1) clinically atypical cases, (2) families without a clear-cut X-linked pattern of inheritance, and (3) families with affected male and female siblings, suggesting an autosomal recessive form of muscular dystrophy. If the Western blot demonstrates dystrophin deficiency, the symptoms may be due to a point mutation or subexonic rearrangements. If dystrophin immunostaining and Western blot analysis are normal, another muscular dystrophy (e.g., sarcoglycanopathy) must be considered.

Females with Dystrophinopathy

Female patients can have an early-onset, progressive muscular dystrophy if they have the following: (1) 45X0, 46XY, or Turner mosaic karyotypes; (2) an apparently balanced X/autosome translocation with breakpoints in Xp21, within the dystrophin gene, and preferential inactivation of the normal X; and (3) a normal karyotype but nonrandom (skewed) X chromosome inactivation leading to diminished expression of the normal dystrophin allele. Therefore, following the exclusion of other neuromuscular diseases (e.g., polymyositis, spinal muscular atrophy) by EMG and muscle biopsy, chromosomal analysis should be considered in all symptomatic females, especially those with highly elevated serum CK levels. Further study with a DNA deletion test may be ­diagnostic in a symptomatic female, especially in cases with 45XO, 46XY, or Turner mosaic karyotypes. About 2.5 to 10 % of manifesting carriers may have clinically apparent muscle weakness. Almost all symptomatic female carriers of mutant dystrophin genes show skewed X-inactivation, which can be detected by a PCR-based androgen receptor assay.

Immunohistochemical stains in muscle biopsies from manifesting carriers with significant elevation of CK levels generally show high proportions of dystrophin-negative fibers, creating a mosaic pattern of dystrophin immuno­fluorescence. In asymptomatic carriers with increased CK, there may be dystrophin-negative fibers by IF; however, if the CK levels are normal, dystrophin-negative fibers are difficult to detect.

Genetic Counseling

With clear X-linked transmission, genetic counseling is similar to that for all X-linked recessive diseases. However, mothers of children with DMD or BMD should be made aware of the rare risk of mild skeletal muscle weakness or dilated cardiomyopathy in carriers, for the purpose of an adequate follow-up evaluation of themselves and their daughters.

In sporadic cases, with a negative mutation analysis in the mother of the proband, the parents should understand that germline mosaicism cannot be excluded; because of its relatively high incidence (20 %), there will be at least a 7–10 % statistical risk (may be higher in individual cases) of recurrence of DMD/BMD in subsequent male offspring. Again, because of possible germline mosaicism, sisters of a sporadic case should be tested for carrier status also, even if the mother of the sporadic proband is DNA deletion negative.

Treatment and Management

Supportive Treatment

The main goal in the management of DMD/BMD is to maintain ambulation for as long as possible and manage the associated complications, such as joint complications, scoliosis, cardiomyopathy, respiratory insufficiency, and weight gain. Therefore, therapeutic interventions in DMD/BMD patients require a multidisciplinary approach, aimed at maintaining function, management of pulmonary and cardiac complications, improving quality of life, and providing psychological support.

Contractures begin early; daily passive stretching exercises to minimize and delay contractures of iliotibial bands, Achilles tendons, and hip flexors are the mainstays of physical therapy. Lightweight plastic ankle-foot orthoses (AFOs) can be applied during sleep to prevent equinus contractures. Contractures of shoulders and elbows are usually not a major functional problem because of the patient’s inability to abduct the arms or fully extend the elbows at advanced stages of the disease. Contractures of wrists and fingers should be treated by passive mobilization and by wearing wrist orthoses at night [71]. Exercise during physical therapy should be limited, especially if it induces muscle pain.

By the age of 9 years, standing and walking can be maintained by using lightweight plastic ankle-foot orthoses or long leg braces (knee–ankle–foot orthoses, KAFOs), the ­latter usually in conjunction with a walker to help maintain balance [151]. The fitting and use of KAFOs may involve and orthopedic surgical intervention (Achilles tendon release) and intense physical therapy. The use of a standing frame should be introduced before the child is no longer able to walk. A standing frame used for a few hours a day, even with minimal weight bearing, can be important in preventing and reducing the severity of contractures, decubitus ulcers, and scoliosis and also improves bone density and gastrointestinal and respiratory functions.

Surgical release of contractures of the hip flexors, iliotibial bands, and Achilles tendons combined with the use of braces has been demonstrated to prolong the ability to walk by 1–3.5 years [72, 151–158]. Although this is a relatively short period of time, it can be functionally and psychologically very important for the patient and his family [159]. Furthermore, it appears that prolongation of ambulation, or even standing, delays the onset of scoliosis [158]. Several combined or isolated surgical procedures and techniques, such as release of tensor fasciae latae, tenotomy of the Achilles tendon, and posterior tibial tendon transfer, are used to obtain these results. However, timing of the surgical procedures, the type of operation, and the need for postoperative bracing are the subject of considerable debate in the literature [159]. Controversy has focused on the potential benefit of hip abduction contractures, which provide a more stable broad-based gait, and the need to correct them [159]. The current approach in many centers is strictly individualized and some patients elect not to have surgery. Markedly weak hip and quadriceps muscles are often deterring factors from surgery since reambulation can be difficult even if the knees and feet are straight. It is preferable to operate on patients while they are still ambulatory, because recuperation is easier and there is less need for postoperative bracing. Patients still ambulating with isolated equinus contractures often benefit from heel cord lengthening with posterior tibial tendon transfer [151, 159].

Scoliosis inevitably happens with wheelchair confinement. In order to avoid asymmetric positioning, the wheelchair should be properly fitted with a reclining back and neck extender and lateral chest wall supports, which may retard the development of scoliosis. During the ambulatory phase a spinal radiography is warranted if scoliosis is observed clinically and is indicated as a baseline for all patients around the time that wheelchair dependency becomes evident. Monitoring with an anteroposterior spinal radiography is recommended yearly for curves less than 15°–20° and every 6 months for curves more than 20° irrespective of steroid treatment up to skeletal maturity [160]. Surgical correction of scoliosis should be considered when the spine is at Cobb angle between 20° and 40° and should be planned before respiratory and cardiac functions decline significantly. To decrease the operative risk, it is optimally performed when FVC is still >30 % [160, 161]. The main reason for anterior spinal fusion is to prevent further progression of spinal ­deformity. Long-term objectives of the surgery are to achieve a good sitting posture, comfort, and quality of life by avoiding the complications of progressive scoliosis [160]. Cardiac and pulmonary evaluations should always precede surgery. Adverse anesthetic reactions during surgery can be minimized by the use of appropriate agents [162]. Boys who have been treated with daily corticosteroids have a greatly decreased risk of scoliosis [163]. Thoracic-lumbar-sacral orthoses are not very helpful in the prevention or arrest of evolving scoliosis [157, 164–169].

Weight control is important and should be monitored carefully. As mobility declines, weight gain is common and contributes to the loss of ambulation. Dietary consultations are recommended in patients who are receiving prednisone therapy to ensure an adequate intake of vitamin D and calcium, provide guidance regarding weight control, plan a healthy diet, and monitor calorie and sodium intake.

Symptomatic involvement of gastrointestinal smooth muscle is more common in older patients and may lead to constipation, impaction, and even acute gastric dilatation. Care givers should be encouraged to promote a diet rich in fiber and adequate hydration of patients. Vaccinations against influenza and pneumococcal infections should be performed regularly and can be administered to patients treated with corticosteroids.

After the age of 6 years or older, pulmonary function studies should be performed yearly. In nonambulatory patients, respiratory assessment may become necessary during clinical visits at least every 6 months. During nonambulatory stage, overnight monitoring of oxygen saturation with pulse oximetry allows the detection of early nocturnal hypoventilation and is recommended at least once every 6 months [170]. Nocturnal hypoventilation responds well to initiation of noninvasive intermittent positive pressure ventilation. Daytime lung function parameters predict sleep hypoventilation [171] and may prove to be useful in appropriate scheduling of polysomnography and noninvasive ventilation during sleep. Respiratory assistance may be used during periods of respiratory infections.

Baseline assessment of cardiac function is recommended at the diagnosis or by the age of 6 years and the care team should include a cardiologist who should be involved in diagnosis and management of cardiomyopathy. A minimum cardiac assessment to include EKG and echocardiogram is recommended once every 2 years until the age of 10 [170]. Annual cardiac assessments could begin at the age of 10 or at an earlier age if abnormalities are detected. Cardiac transplantation is a therapeutic option that can be lifesaving for patients with Becker MD assuming that they have first had a careful trial of medical therapy and, in some, implantable cardiac defibrillators [102, 172, 173].

Anesthesia must be approached with caution in patients who have dystrophinopathies [174]. Patients with DMD and BMD can have severe complications from anesthesia, ­including cardiac arrest. Most complications seem to be related to use of succinylcholine, a muscular relaxant that may trigger hyperkalemia [174]. Others have been attributed to use of volatile anesthetic agents. Patients also can have a reaction similar to malignant hyperthermia [174], develop rhabdomyolysis, and have masseter muscle spasm.

Pharmacologic Treatment

Corticosteroids are the best treatment option currently available. Randomized control trials have shown that oral prednisone produces a significant increase in muscle strength, pulmonary function, and overall functional ability in DMD [175, 176]. The mechanism of improvement seen in individuals with DMD treated with prednisone is not well understood but thought to be through an anti-inflammatory effect related to reduction in total T cells and cytotoxic–suppressor T cells [175]. This improvement is most effective with a single daily dose of 0.75 mg/kg regimen, begins within 10 days, and reaches a plateau after three months. Observed side effects include weight gain, hypertension, behavioral changes, growth retardation, and cataracts [175, 176]. There is also an increased frequency of vertebral and long bone fractures with prolonged corticosteroid use [163]. Deflazacort, a synthetic derivative of prednisolone (0.9 mg/kg/day), appears to be equally effective but is not available in the United States [177]. Daily corticosteroids improve muscle strength and function, significantly slow the progression of weakness, prolong ambulation to the mid teens or later, and delay the onset of respiratory and cardiac dysfunction [178, 179]. Asymptomatic cataracts are more common with deflazacort, and weight gain is more prominent with prednisone. If adverse effects require a reduction in prednisone dosage, doses as low as 0.3 mg/kg/day produce improvement that is less prominent but still significant. High-dose weekly prednisone, 5 mg/kg, given each Friday and Saturday, can be considered as an alternative to daily treatment in males on a daily regimen with excessive weight gain and behavioral issues [180].

Data regarding the optimal age to begin treatment with corticosteroids or the optimal duration of such treatment are insufficient. Thus, at this point corticosteroid therapy remains the treatment of choice for affected individuals between ages 5 and 15 years. It is recommended that boys with DMD who are older than age 5 years should be offered treatment with prednisone (0.75/mg/kg/day, maximum daily dose: 40 mg) as soon as plateauing or decline in motor skills is noted. Prior to the initiation of therapy, the potential benefits and risks of corticosteroid treatment should be carefully discussed with parents and caregivers individually.

Information about the efficacy of prednisone in treating individuals with BMD is limited. Many clinicians advocate continuing treatment with glucocorticoids after loss of ambulation for the purpose of maintaining upper limb strength, delaying the progressive decline of respiratory and cardiac function, and decreasing the risk of scoliosis. Retrospective data suggest that long-term daily corticosteroid treatment may reduce the progression of scoliosis; however, an increased risk for vertebral and lower limb fractures has been documented [163].

Emerging Therapies

Although there is no curative treatment for DMD, there are many areas of therapeutic intervention under investigation. Three major treatment strategies have emerged to repair the DMD gene defect which include viral-mediated gene replacement, exon skipping, and mutation suppression. Additional strategies include but are not limited to utrophin upregulation, muscle enhancement, fibrosis reduction, and nNOS modulation which will be discussed in brief.

Gene Replacement

Adeno-associated virus (AAV) delivery remains the most promising strategy for replacement of the dystrophin gene. It is nonpathogenic and remains stable in nonreplicating cells (i.e., muscle), and multiple serotypes exhibit tropism for muscle. One caveat for AAV delivery as a treatment for DMD is the large size of the dystrophin gene which exceeds the packaging capacity of AAV (<5kb). Fortunately, the modular structure of dystrophin allows some flexibility; deletions of nonessential coding regions allow dystrophin to retain significant function if the reading frame is intact. This conclusion was initially based on a clinical observation in a BMD patient with a large in-frame deletion of exons 17–48 removing a significant portion of the rod domain [181]. The patient remained ambulatory until age 61 despite the absence of 46 % of the dystrophin gene. This led to the design of mini- and micro-dystrophin transgenes that were able to fit into AAV. Numerous proof-of-principle studies using AAV delivery of miniature dystrophin genes have shown reversal of the dystrophic phenotype in the mdx mouse model for DMD. The first trial of AAV-mediated delivery of mini-dystrophin defined potential hurdles to consider for future trials that restore dystrophin regardless of approach [182]. These lessons include the potential for an immune response generated against novel epitopes presented by exogenous dystrophin in patients with large endogenous deletions as well as immunity primed by revertant fibers. Revertant fibers result from spontaneous second-site mutations which restore the reading frame of dystrophin. Once thought to be tolerizing, the AAV mini-dystrophin trial revealed that revertant fibers could be immunogenic and accelerate responses following gene transfer.

Upregulation or replacement of utrophin is another therapeutic strategy for DMD that has shown promise. Utrophin shares 80 % sequence homology with dystrophin and has been shown to partially restore function as a dystrophin surrogate in preclinical transgenic mice [183] or gene replacement studies [184]. Utrophin expression is limited to the neuromuscular and myotendinous junctions in normal muscle. However, in both dystrophic mice and DMD patients, it is overexpressed in the sarcolemma of all muscle fibers, partially compensating for the mechanical role of dystrophin in the membrane. Upregulation of utrophin holds particular advantage because of the unlikely occurrence of an immune response as seen following mini-­dystrophin gene replacement [182]. Alternative strategies have also emerged to upregulate utrophin at the sarcolemma including several small molecules which demonstrate transcriptional upregulation through the utrophin-A promoter [185–187] or by the use of DAP-stabilizing molecules such as biglycan (rhBGN) [188].

Exon Skipping

Exon skipping is a second molecular treatment approach which is targeted at the messenger RNA (mRNA) level allowing one or more exons to be omitted to restore the dystrophin reading frame. This is accomplished with antisense oligonucleotides (AONs) that are artificially synthesized to hybridize in a complementary fashion to mRNA to modify splicing. Preclinical efficacy has been demonstrated in the mdx mouse, dystrophin/utrophin knockout mouse, and CXMD dog [189–191]. It has been predicted that through targeted skipping of particular exons, as many as 60–80 % of DMD mutations could be corrected. Two phase I safety trials were conducted in DMD patients targeting exon 51 using AONs with two different chemical backbones, 2-O-methyl AON-PRO051 and phosphorodiamidate morpholino oligomer (PMO) – AVI-4658 [192, 193]. Safety was demonstrated in both studies which were limited to an intramuscular injection of the AON in a single muscle. Phase I/II extension studies were performed with both AONs to assess efficacy and tolerability following systemic delivery. In the PRO051 trial, dose-related efficacy was achieved with evidence of new dystrophin expression in approximately 60–100 % of muscle fibers in 10 of 12 patients and modest improvement in the 6-min walk test [194]. In the AVI UK phase II open-label study with AVI-4658 (Eteplirsen), 7 of 19 patients saw a modest response with a mean increase of sarcolemmal dystrophin from 8.9 % to 16.4 % [195]. Additional phase II randomized, double-blind, placebo-controlled trials are under way to assess multiple-dose efficacy.

Mutation Suppression

A second molecular approach involves suppression of stop codon mutations of the DMD gene that comprise approximately 15 % of DMD cases. Two pharmacologic tactics have shown preclinical efficacy and have also been tested clinically. In mdx mice, in vivo mutation suppression was shown with the aminoglycoside antibiotic, gentamicin [196]. In the most definitive trial, DMD patients (n = 16) with stop codons, treated weekly or twice weekly for 6 months (7.5 mg kg IV), showed a significant increase in dystrophin levels with the highest levels reaching 13 % and 15 % of normal. Muscle strength was stabilized and a modest increase in forced vital capacity was achieved. Although this study demonstrates the therapeutic potential of gentamicin, higher doses might be necessary to improve functional outcomes. The known renal toxicity of aminoglycoside antibiotics and the nuisance of intravenous administration have pushed the field to identify an orally administered agent.

Ataluren, formerly referred to as PTC124 (PTC therapeutics), demonstrated promise as an orally administered pharmacologic read-through agent for stop codon mutations [197]. Preclinical studies in the mdx mouse revealed dystrophin expression in skeletal, cardiac, and diaphragm muscle and protected skeletal muscle from eccentric contraction-­induced injury. A phase I study in healthy volunteers established safety and tolerability at doses exceeding what was required for preclinical efficacy [198]. Dystrophin appeared to increase posttreatment in a phase IIa proof-of-concept 28-day study in DMD/BMD patients. Following these results, a randomized, double-blind, placebo-controlled phase IIb trial was conducted evaluating safety and efficacy over a 48-week treatment period. PTC, Inc., released preliminary results indicating a very strong safety profile; however, the primary endpoint of the 6-min walk test did not reach statistical significance [199]. Ataluren is under continued development for the treatment of cystic fibrosis [200] and hemophilia (clinicaltrials.gov NCT00947193); positive results from these studies may provide evidence for further pursuit as a treatment option for DMD.

Muscle Growth Products

Increasing muscle fiber size and strength is another treatment strategy for DMD under intense investigation. Inhibition of the myostatin pathway shows promise for clinical application. Myostatin is a member of the transforming growth factor beta (TGF-β) family and is a potent regulator of muscle growth. Accordingly, myostatin knockout mice demonstrate dramatic muscle hypertrophy and hyperplasia [201]. The role of myostatin as a negative regulator of muscle mass is highly conserved across species including humans, as shown by the identification of a myostatin splice-site mutation leading to the loss of myostatin protein in a hypermuscular family [202]. As a disease characterized by progressive muscle loss, DMD is a natural therapeutic target for myostatin blockade. In one approach, a recombinant human antibody (MYO-029) that binds with a high affinity to myostatin and inhibits its activity [203] was shown to increase muscle mass in immunodeficient mice by approximately 30 % over 3 months, similar to the biological response demonstrated for other myostatin-­neutralizing antibodies [204]. MYO-029 was subsequently studied in a double-blind randomized clinical trial in Becker, limb-girdle muscular dystrophy (including multiple types), and facioscapulohumeral muscular dystrophy where safety but not clear efficacy was established [203]. Based on this principle, a potentially more potent agent is follistatin, a myostatin inhibitor that has been demonstrated to lead to muscle growth in vivo. Delivery of the follistatin gene by adeno-­associated virus (AAV) in mice or nonhuman primates shows dramatic increases in muscle size and strength, and this approach is poised for gene therapy [205, 206].

Apart from myostatin inhibition, insulin growth factor-1 (IGF-1) treatment is another strategy to increase muscle mass and strength under investigation for DMD. IGF-1 is a growth factor and key mediator of anabolic pathways in muscle which stimulates whole body protein metabolism [207]. It is involved in muscle repair and regeneration by stimulating the proliferation and differentiation of skeletal muscle cells [208–210]. Preclinical studies found that transgenic overexpression of IGF-1 in DMD mice increased skeletal and diaphragm muscle mass, increased force generation, and reduced fibrosis and myonecrosis [211, 212]. IGF-1 is already FDA approved for severe primary IGF deficiency. A prospective, randomized, open-label, controlled phase II clinical trial of recombinant IGF-1 (INCRELEX™) has been initiated in glucocorticoid (GC)-treated DMD patients to test its ability to preserve muscle function.

Several other therapeutic strategies are also on the horizon for DMD in either proof-of-principle animal studies or in clinical trial. Fibrosis is a confounding factor for any treatment for DMD. Proliferation of endomysial connective tissue limits muscle regeneration, contributing to progressive muscle weakness. Strategies to prevent or reduce fibrosis alone or in combination with other therapies are under exploration including TGF-β blockade and altering expression of micro-RNAs [213, 214]. Another therapeutic target is neuronal nitric oxide synthase (nNOS). Two variants of nNOS contribute to normal muscle metabolism by attenuating vasoconstriction and helping maintain the ability of contracting muscle to generate force following exercise. nNOS is either downregulated or mislocalized in dystrophic tissue [215] and contributes to overall disease pathology; therefore, reversal of ischemic-induced pathology by upregulating nNOS expression has potential as a treatment strategy. To ­summarize, multiple therapeutic strategies are on the horizon for treating dystrophinopathies with the goal of replacing the missing dystrophin gene or reversing resultant pathology. This multifaceted disease may not be curable by any one therapy as demonstrated by various treatments under investigation presented here.