Historical Background

Duchenne’s description of DMD was published in the 1861 edition of his book on the electrical stimulation of muscle, but John Little and Edward Meryon had first reported this entity 8–9 years earlier. In fact, in 1852, Meryon described very accurately the clinical course of the disease in four brothers and also the pathological findings on microscopic examination of muscle. In 1853, Little described two brothers with “abnormal increase of bulk of muscle combined with contraction and adipose degeneration,” who were unable to walk after the age of 11 years.

In 1861, Duchenne described the disease as “hypertrophic paraplegia of infancy due to a cerebral cause” but, by 1868, he recognized that it was muscular in origin and was the first one to propose criteria for diagnosis. He also used a small harpoon to perform muscle biopsies and described the replacement of muscle by fat and connective tissue.

The occurrence of a more benign form of X-linked muscular dystrophy was first proposed in 1955 by Becker and Kiener and Walton ; the disorder was named Becker muscular dystrophy after several families were reported with similar features. It was not clear at that time whether DMD and BMD were genetically distinct disorders, or allelic forms of the same genetic entity.

In the early 1980s, advances in cytogenetics and molecular genetics led to mapping of the gene responsible for DMD in band 21 of the short arm of the X chromosome (Xp21). First, a number of female patients with muscular dystrophy were reported who, on cytogenetic analysis, had X;autosome translocations (e.g., X;11, X;21); the translocation breakpoint in the X chromosome was consistently at Xp21. Second, using restriction fragment length polymorphisms (RFLPs) and linkage analysis, cloned DNA segments were linked to the DMD locus on the short arm of the X chromosome, supporting the Xp localization of the DMD gene. Third, in 1985, Francke et al. at Yale University detected a microscopically observable deletion at Xp21 on the short arm of the X chromosome in a male patient (“BB”); this patient suffered from Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and the McLeod syndrome. Subsequently, in 1985, by using a competitive hybridization strategy, Kunkel et al. at Boston Children’s Hospital isolated specific DNA fragments absent from the genome of patient “BB” ; molecular analysis of a large number of DNAs from DMD patients showed that one of these fragments/clones was deleted in 6.5% of DMD patients. A 200 kilobase (kb) chromosomal walk was initiated from this clone which, in 1986, led to the isolation of an exon of the DMD gene and partial cloning of a muscle-specific transcript. Using a different strategy, Worton’s group at the University of Toronto isolated a breakpoint junction clone from the DNA of a female patient with an X;21 translocation ; with starting point this junction clone, in 1987 a 180 kb walk led to the identification of a second exon of the DMD gene and more of the cDNA of the 14 kb transcript. In 1987–88, the complete cDNA sequence of the DMD gene was cloned and sequenced by Kunkel’s group and the protein product was named dystrophin. Dystrophin was localized in the sarcolemma of muscle fibers and was shown to be almost completely absent in DMD and decreased in BMD. These major scientific advances have made diagnosis and genetic counseling far more accurate and have allowed reliable prenatal testing; they have also paved the way for the discovery of effective therapies.

DMD Gene

DMD and titin are the largest genes yet identified in humans. The DMD gene, with a 14 kb transcript and 79 exons, spans approximately 2.2 megabases on the short arm of the X chromosome (Xp21.3-p21.2), or about 1% of the entire X chromosome. The introns of the gene are large, with some near the 5′ third of the gene and between exons 44 and 45 being nearly 200 kb. The genomic (exonic and intronic) sequence of the DMD gene can be obtained from the National Center for Biotechnology Information (NCBI) / Genbank web site pages and also from the Leiden Muscular Dystrophy web pages. The DMD gene product dystrophin is recognized on Western blots of human skeletal muscle proteins using antidystrophin antibodies. With the use of immunocytochemistry, dystrophin has been localized to the cytoplasmic side of the plasma membrane of muscle fibers. Dystrophin has four functional domains: the amino-terminal or actin-binding domain, the rod domain, the cysteine-rich domain and the carboxy-terminal domain.

• •
  

  The amino-terminal domain (exons 1–8) binds to actin through three high-affinity actin-binding sites.

  
  
• •
  

  The rod domain (exons 9–63) is large, comprised of 24 homologous “spectrin-like” repeats forming an α-helical structure. The repeats are interrupted by two nonhelical regions known as “hinges,” which are thought to confer flexibility on the rod domain during muscle contraction.

  
  
• •
  

  The cysteine-rich domain (exons 64–69) is a region of the DMD gene near the carboxy-terminus that seems to be the region of dystroglycan binding.

  
  
• •
  

  The carboxy-terminal domain (exons 70–79) binds with a protein complex that links the cytoskeleton with membrane proteins that in turn bind with proteins in the extracellular matrix.

Therefore, dystrophin is part of a large, tightly associated glycoprotein complex containing other proteins, the so-called dystrophin associated proteins (DAP) ( Figure 30.1 ).

The dystrophin associated protein complex and other proteins known to be involved in muscular dystrophy. Arrows indicate the protein components mutated in various muscular dystrophies. Alterations in the dystrophin gene cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Mutations in the sarcoglycan proteins, caveolin-3 and dysferlin, lead to forms of limb girdle muscular dystrophy (LGMD). Laminin α2-chain is mutated in a subtype of congenital muscular dystrophy (CMD) without structural brain anomalies, as is α7-integrin. Abbreviations: COOH, carboxy-terminal domain; CR, cysteine-rich domain; HH, two helices of the coiled-coil domain; MM, Miyoshi myopathy; NH ₂ , amino-terminal domain; Syn, syntrophin-binding domain.

Courtesy of K. O’Brien, C. Bönnemann, and L. Kunkel, Boston Children’s Hospital, Boston, MA, USA.

The DMD gene can generate several cell type-specific isoforms of dystrophin of different molecular weight, each driven by a distinct promoter. These promoters drive the transcription of the dystrophin gene from their own first exon and give rise to dystrophin isoforms of various molecular weights ( Table 30.1 ) ( Figure 30.2A, B ). The brain, muscle, and Purkinje cell dystrophins are predicted to have a molecular weight of 427 kilodaltons (kDa), whereas the glial/general and Schwann cell dystrophins are encoded by carboxy-terminal transcripts and have predicted molecular weights of 71 and 116 kDa; they are designated as B/Dp427, M/Dp427, P/Dp427, G/Dp71, and S/Dp116 dystrophins, respectively. The dystrophins of retina (R/Dp260) and brain/kidney (B-K/Dp140) have been described also ( Figure 30.2B ) .

( A ) Dystrophin gene structure and promoters. The schematic diagram shows 7 distinct promoters, which drive the transcription of the dystrophin gene from their own first exon and give rise to dystrophin isoforms of various molecular weights. It also shows a schematic map of 79 exons of the dystrophin gene, protein domains (boxes), plus the three 5′ promoter-specific first exons in red (Dp427B-M-D). Lines in blue indicate the 3′ promoters and the first exons of isoforms: Dp260 (retina), Dp140 (brain/kidney), Dp116 (Schwann cell), Dp71 (general). Exons in green are alternatively spliced or skipped. ( B ) Schematic diagram of three full-length dystrophins and smaller isoforms. The actin-binding domain (red/pink) is shared among a number of the various dystrophins, utrophin, α-actinin, and β-spectrin. The internal rod-domain (green) consists of homologous repeats of ~109 amino acids. The carboxy-terminus (yellow) is homologous only to utrophin (gold). Abbreviations: M, muscle; B, brain; P, Purkinje cell; R, retina; B/K, brain/kidney; S, Schwann cell; G, general or glial; DRP, dystrophin related protein; NH ₂ , amino-terminus; COOH, carboxy-terminus; CYS, cysteine-rich domain.

(A) is from Ferlini et al. Used with permission.

Other dystrophin variants (i.e. alternatively spliced transcripts) result from alternative splicing of exons 71, 74, 78, and 79 and are expressed in muscle, brain and heart tissue; alternative splicing occurs in regions involved in binding of dystrophin to the DAP complex and, therefore, may regulate their association in various tissues. Muscle dystrophin is expressed in skeletal (all muscle fiber types), smooth, and cardiac muscles, and also in the outer plexiform layer of the retina. The expression of this and probably other isoforms is developmentally regulated; dystrophin is first detected in human fetal muscle at 9 weeks of gestation and its expression is increased as myoblasts differentiate into multinucleated myotubes. The highest expression of brain dystrophin is in the neocortex and hippocampus; Purkinje cell dystrophin comprises most or nearly all of cerebellar dystrophin. Dp71 is expressed in glial cells, viscera, and mature cardiac and fetal skeletal muscle but not in mature skeletal muscle. While muscle dystrophin is detected on the plasma membrane of skeletal muscle fibers, it appears to be particularly abundant in neuromuscular and myotendinous junctions.

The different isoforms must be interacting with different proteins in the various tissues in which they are expressed. In DMD, the most important of these isoforms are the three full-length, large molecular weight (427 kDa) dystrophins, present in skeletal and smooth muscle (muscle type, Dp427), in cerebral cortex and hippocampus (brain type, Dp427), and in Purkinje cells (Purkinje type, Dp427).

The Dystrophin Associated Protein Complex

As discussed above, the dystrophin-associated proteins together with dystrophin form a complex known as the dystrophin associated protein complex (DAP), which, acting as a bridge, connects the intracellular cytoskeletal actin to the basal lamina through the extracellular matrix ( Figure 30.1 ). The DAP complex is believed to stabilize the sarcolemmal membrane during contraction and relaxation. The extracellular matrix is a network situated between the sarcolemma and the extracellular basal lamina and is composed mainly of laminin, collagen, fibronectin, and proteoglycans. The DAP complex will be discussed herein briefly and in detail in Chapter 34 on Limb-girdle Muscular Dystrophies.

The DAP complex is composed of three groups of proteins:

• 1.
  

  Dystroglycan group

  
  
  
  
  • 
    

    α-dystroglycan

    
    
  • 
    

    β-dystroglycan.

  
  
  
  
• 2.
  

  Sarcoglycan group

  
  
  
  
  • 
    

    α-sarcoglycan (or adhalin)

    
    
  • 
    

    β-sarcoglycan

    
    
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    γ-sarcoglycan

    
    
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    δ-sarcoglycan

    
    
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    sarcospan.

  
  
  
  
• 3.
  

  Syntrophin/dystrobrevin group

  
  
  
  
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    α-syntrophin

    
    
  • 
    

    β1-syntrophin

    
    
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    β2-syntrophin

    
    
  • 
    

    α-dystrobrevin

    
    
  • 
    

    β-dystrobrevin.

  
  

The dystroglycan subcomplex functions as a receptor of certain extracellular matrix proteins. Indeed, α-dystroglycan is an extracellular protein that binds to β-dystroglycan, which is located in the sarcolemma, and to laminin-α2 in the extracellular matrix. β-dystroglycan is a transmembranous protein that binds intracellularly to dystrophin (at the cysteine-rich domain and the first half of the carboxy-terminal domain).

The “dystrophin-axis” (actin-dystrophin-dystroglycan-laminin) serves a very important function in creating a link between the sarcolemma and the extracellular basal lamina, and thus forms the structural basis for mechanical stabilization of the sarcolemma during contraction and relaxation of muscle fibers.

Laminins are a group of proteins situated in the basal lamina and extending into the extracellular matrix. They are each a heterotrimer molecule composed of 1 heavy chain (α2) and 2 light chains (β1 or β2 and γ1). Several α, β and γ chains have been identified, and their various combinations result in different types of laminin. Merosin is a term that encompasses all laminins that contain an α2 heavy chain. Laminin α2-chain mutations have been described in a subset of patients with congenital muscular dystrophy (discussed in Chapter 29 ). In the sarcolemma, several proteins have been considered as receptors for laminins, including dystroglycans (described above), integrins (α7-β1 integrin), and lectins.

The sarcoglycan group is comprised of a number of sarcolemmal transmembrane proteins (α-, β-, γ-, δ-sarcoglycans and sarcospan) and is described as the side arm of the dystrophin axis because of its lateral linkage with the dystrophin-dystroglycan complex. However, the exact details of the dystrophin-sarcoglycan complex association have not yet been elucidated. Deficiency of the various sarcoglycan complex proteins is responsible for several limb-girdle muscular dystrophies, known as sarcoglycanopathies (discussed in detail in Chapter 34 ). A muscle-specific form of filamin, termed filamin 2 (FLN2, FLNC or γ) was identified as a γ- and δ-sarcoglycan interacting protein. FLN2 is located in two intracellular pools, with about 97% of the cellular content contained in the cytoplasm and approximately 3% localized at the sarcolemmal membrane. Interestingly, in LGMD and DMD patients the membrane content of FLN2 increases to about 30%, which suggests that FLN2 binds to membrane-bound proteins other than sarcoglycans, possibly β1-integrin; the increase of FLN2 at the membrane in patients with LGMD and DMD and its diminished translocation intracellularly to bind F-actin also imply an essential role in maintaining membrane integrity.

The second group of dystrophin-associated proteins contains the intracellular syntrophins and the dystrobrevins. The syntrophin subcomplex, made of 3 intracytoplasmic proteins (α-, β1-, and β2-syntrophin), also binds to the carboxy-terminus of dystrophin. In addition, the syntrophins have been shown to bind neuronal nitric oxide synthase (nNOS). Dystrobrevin is another intracytoplasmic protein complex, comprised of α- and β-dystrobrevin, that binds to syntrophins and dystrophin. The dystrobrevins, in addition to binding to dystrophin, are highly homologous to the dystrophin carboxy-terminal domain. The quantity of dystrobrevins is reduced in both DMD and patients with sarcoglycanopathies, probably on a secondary basis.

DMD Gene Mutations

Of the DMD/BMD mutations identified thus far, most are deletions, detected in approximately 50–65% of males with DMD and 65–70% of males with BMD. Partial gene duplications have also been reported in a small percentage of patients with DMD and BMD (about 5–10%). Deletions or duplications involve 1 or more exons of the DMD gene. In both DMD and BMD, partial deletions and duplications cluster in two recombination hot spots, one proximal at the 5′ end of the gene in the region of exons 3–7 (nearly 30%) and one more distal, comprising exons 44–53 (nearly 70%), with additional deletions throughout the rest of the gene. The hot spots seem to occur in the regions of the gene where the introns are long; for example, as discussed earlier, the intron 44 between exons 44 and 45 is extremely large; the genomic breakpoints of the 5′ hotspot lie within introns 2 and 7. In the remaining 25–35% of DMD patients without detectable deletions or duplications, and in about 20–30% of males with BMD, the molecular lesions represent single base changes, small deletions or insertions, or splicing errors.

Nonsense mutations occur more commonly in DMD than in BMD, in the range of 20–25% of DMD cases compared with fewer than 5% of BMD cases. In both DMD and BMD, a substantial portion of sequence changes are splice site mutations and small insertion/deletion mutations (in/del). In neither DMD nor BMD are missense mutations a common cause. Atypical mutations (deep intronic, those rarely occurring in the 5′ and 3′ untranslated regions) account for less than 1% of all DMD gene mutations.

Published studies have failed to reveal any apparent correlation between the size of dystrophin gene deletions and the severity and progression of the DMD/BMD phenotype. The molecular basis of Duchenne versus Becker muscular dystrophy seems to be related to the disruption or preservation of the amino acid translational reading frame by the deletion mutations ( Figure 30.3 ). In other words, during the synthesis of mature mRNA, the joining ends of the exons (after splicing of introns) have to be in phase in order to maintain the correct translational open reading frame. A deletion that juxtaposes exons that shift the translational reading frame (out-of-frame deletion) usually results in unstable mRNA and eventually in the production of a severely truncated dystrophin molecule, which is rapidly degraded in the cell and, thus, leads to a more severe DMD phenotype. This phenomenon is known as nonsense mediated decay and aims to eliminate the pathological allele to avoid dominant negative effects. Other mutations disrupting the reading frame include stop (nonsense) mutations, some splicing mutations, and duplications. A deletion which juxtaposes exons that preserve the translational reading frame (in-frame deletion) will result in an internally deleted but semifunctional dystrophin protein (with intact amino- and carboxy-termini), which can persist at some quantity in the cell and thus result in a milder BMD phenotype. Other “in-frame” mutations include duplications, some splicing mutations and most non-truncating single base changes. However, exceptions to this “reading frame hypothesis” occur in approximately 8% of cases ; they were initially reported by Malhotra et al. and subsequently were noted by others as well.

Molecular basis for Duchenne muscular dystrophy (DMD) versus Becker muscular dystrophy (BMD) phenotypes. The top of the figure indicates the Xp21 map position of DMD/BMD locus and also approximates the size of the DMD gene, its transcription product (mRNA), and dystrophin protein. The bottom of the figure shows how a small deletion can lead to a severe DMD phenotype, in contrast to a large one, which yields a milder BMD phenotype. On the left, deletion of single exon (crossed-out box) disrupts the translational reading frame because exons flanking the deletion are out of phase (out-of-frame deletion). Because mRNA can be translated only in triplet codons, T-GAT… will be converted to TGA-T…, which creates a nonsense mutation (TGA codon is translation stop), leading to severely truncated dystrophin protein. On the right, a relatively larger deletion contains integral number of triplet codons and therefore preserves the reading frame (in-frame deletion). The resulting protein is internally deleted and probably semifunctional, accounting for the milder BMD phenotype.

From Hoffman and Kunkel LM. Reprinted by permission of Cell Press; © Cell Press.

When in-frame deletions in BMD patients are compared, as a general rule some regions of the dystrophin protein seem more critical than others; deletions in the regions coding for the amino-terminal or carboxy-terminal domains of dystrophin result in more severe phenotypes than mutations affecting the rod domain ; nonetheless, exceptions to this rule do occur also and are discussed below. Exceptions most frequently occur with deletions of exons 3 to 7 or exon 45; the exceptions are also detailed below. Although these mutations are out-of-frame they can result in BMD, DMD, or an intermediate phenotype. Exon-skipping events or activation of new cryptic translational start sites may create situations in which apparently out-of-frame deletions behave as in-frame deletions or vice versa. Alternatively, posttranscriptional mechanisms (e.g. ribosomal frame shifting or reinitiation) may help restore the mRNA reading frame in BMD patients with out-of-frame 3 to 7 exon deletions. In addition, variations in the severity of the phenotype among patients with similar mutations (e.g. deletion of exon 45) have been reported, supporting the contribution of other factors in the phenotypic expression of these mutations. Furthermore, very large deletions or deletions in protein-binding domains (e.g. cysteine-rich domain), even if in-frame, can result in a severe phenotype.

Dystrophin

Dystrophin is a rod-shaped molecule of 427 kDa and can easily be detected on Western blots (immunoblots) of 100 μg of total muscle protein derived from a small portion of a muscle biopsy, by utilizing antidystrophin antibodies to the amino-terminus, rod domain, and carboxy-terminus. The quantity and quality of dystrophin can be evaluated either visually or by using densitometry. If the 427 kDa dystrophin protein is normal in size and amount, the diagnosis of DMD or BMD can be excluded. More than 99% of DMD patients display complete or almost complete absence of dystrophin in skeletal muscle biopsy specimens. Most patients with BMD (about 85%) have dystrophin of abnormal molecular weight, either smaller (80%) or larger (5%), in gene deletion or gene duplication cases, respectively; the dystrophin amount is reduced to normal. About 15% of the BMD patients, however, have normal-sized protein of reduced quantity. The Western blot test is very specific, because patients with neuromuscular diseases other than DMD/BMD have normal dystrophin. Thus, dystrophin immunoblotting, in conjunction with muscle biopsy immunostaining and other clinical and laboratory parameters, can be used to help predict the severity of the evolving muscular dystrophy phenotype ( Table 30.2 ).

Pathogenesis

The functional features of dystrophin are not fully understood and probably differ in the various isoforms present in different tissues. In skeletal muscle the primary role of dystrophin is probably mechanical stabilization of the plasma membrane. Dystrophin-deficient muscle fibers of the mdx mouse exhibit an increased susceptibility to contraction-induced sarcolemmal rupture. Dystrophin may allow the membrane to fold during contraction-relaxation of muscle fibers ; it may also function as a stabilizer of membrane-spanning molecules such as sarcoglycans. Therefore, the clinical manifestations of dystrophin deficiency may be related to the loss of stability of sarcoglycans or of other DAP complex glycoprotein functions. Furthermore, because the sarcolemmal expression of the DAP complex proteins depends on the presence of normal dystrophin, there is almost always significant secondary reduction in the amount of DAP glycoproteins in the muscle tissue of patients with DMD.

Histopathologic findings and animal experiments support the notion that a decreased amount of dystrophin results in a mechanically weakened plasma membrane, which is prone to focal tears during contractile activity. This allows then a massive influx of extracellular calcium, which activates proteolytic enzymes like calpains and thus leads gradually over time to necrosis of muscle fibers. This theory could explain why dystrophinopathies are progressive diseases; it could also serve as an explanation for the fact that small-caliber fibers (i.e. fibers of extraocular muscles) are relatively spared, as the relative mechanical stress per unit surface-membrane area in small-caliber muscle fibers is less than that exerted in larger fibers. The report by Kimura et al. seems to illustrate this theory. They describe a patient having both DMD and X-linked recessive myotubular myopathy (MTM1) whose muscle biopsy demonstrated changes typical of MTM1 only, and lacked the typical myonecrosis of DMD. They hypothesize that not only the immaturity and small size of many fibers, but also the inactivity of muscle fibers due to MTM1 protected these fibers from necrosis.

In mdx mice, despite the increased susceptibility to contraction-induced membrane damage, the severity of muscular dystrophy is quite mild. Several explanations have been postulated.

• 1.
  

  Utrophin expression in muscle, which can compensate physiologically for the absence of dystrophin in mdx mice.

  
  
• 2.
  

  Sialic acid N-glycosylneuraminic acid (Neu5Gc) expression in murine but not in human skeletal and cardiac muscle. It is incorporated in muscle glycoproteins and glycolipids and presumably ameliorates disease expression in mdx mice.

  
  
• 3.
  

  Progressive loss of muscle stem cell function due to shorter telomeres in humans compared to murine muscle stem cells. This may lead to inability to repair the muscle injury induced by dystrophin deficiency in humans.

Novel pathogenetic mechanisms have been described such as impaired signaling, intermittent ischemia of skeletal and cardiac muscle due to vascular smooth muscle dysfunction, and defective membrane repair and resealing of damaged muscle fibers. In particular, filamin proteins (FLN1 and FLN2) have not only been found to be essential in membrane mechanoprotection during force application but also seem to be involved in signal transduction associated with cell adhesion and differentiation and, perhaps, membrane shape change. Thus, the interaction between FLN2 and the sarcoglycans implies a dynamic role of the DAP complex in muscle cell function (i.e. signal transduction) in addition to its originally believed structural role in the maintenance of a link between actin, the sarcolemma, and the extracellular matrix.

Sarcolemmal nNOS catalyzes the production of nitric oxide and it is secured to the sarcolemma by dystrophin, as part of the DAP complex. Nitric oxide mediates vasodilation and thus increases blood flow into muscles, which prevents early muscle fatigue with exercise. Dystrophin deficiency results in loss of muscle nNOS and hence in exercise-induced muscle fatigue.

Utrophin

Utrophin or dystrophin related protein (DRP), a protein that resembles dystrophin in its amino acid sequence, structural organization, and size (395 kDa) ( Figure 30.2B ), is the product of a dystrophin homologous gene mapped to chromosome 6q24. It is present in the cellular membranes of most well-differentiated tissues, intramuscular nerves and blood vessels, Schwann cells, and uterine smooth muscle fibers, and also in myoblasts and myotubes. In skeletal muscle fibers, its expression is confined to the neuromuscular and myotendinous junctions, whereas dystrophin is present throughout the plasma membrane. At the neuromuscular junction, utrophin is detected near the acetylcholine receptor on the crests of the junctional folds, whereas dystrophin is concentrated in the troughs of the junctional folds. During fetal development, utrophin is also expressed in the extrajunctional sarcolemma but at birth it is replaced by dystrophin in this location. As is dystrophin, utrophin is also associated with a glycoprotein complex, similar to the DAP one. Experimental data in the dystrophin deficient mdx mouse suggest that upregulation of utrophin may be able to compensate for dystrophin deficiency. However, it appears that utrophin expression is already upregulated in humans with DMD, resulting in its presence in the extrajunctional sarcolemma. Despite that, modest upregulation of utrophin expression may modify substantially the natural course of DMD. In humans, it would be reasonable to speculate that the spontaneous utrophin upregulation that occurs in DMD is a mitigating mechanism and that in its absence, the rate of progression of the disease would be even faster. This is supported by a report of a young patient with DMD, who had a very severe clinical course, and whose muscle biopsy revealed complete absence of utrophin in addition to absent dystrophin. Also, dystrophin-utrophin double knockout (dko) mice exhibit multiple systemic degenerative changes in addition to premature muscle degeneration. Strategies designed to identify small molecules that could activate the utrophin promoter in humans and thus upregulation of utrophin expression in dystophic muscle, may lead to the discovery of therapies for DMD. Compounds such as biglycan and SMTG1100 upregulate utrophin and reduce the dystrophic pathology and/or symptoms in the mdx mouse.

Germline Mosaicism

Dystrophinopathies are familial diseases; however, sporadic cases do occur in one-third of cases, resulting from a spontaneous de novo mutation in a single germ cell of the mother or grandparent. It can also occur in the proband’s dystrophin gene early in postzygotic embryo development; in that case the mutation will be present in some, but not all, cells of the body, leading to “somatic mosaicism.” If the mother does not have a detectable mutation in DNA derived from her white blood cells but has more than one affected offspring, that may be related to the phenomenon of “germline mosaicism” (i.e. the mutation is present in the germ cells only and is not detectable in a blood sample) ; the latter accounts for 15 to 20% of new DMD mutations. This phenomenon is probably due to a mutation at a mitotic stage of germ cell (egg) lineage development, which results in a subpopulation of mutated eggs; the germ cells derived from the normal progenitor cell(s) will not carry the mutation. The earlier the mutation event occurs in the stages of germ cell lineage development, the higher will be the percentage of mutated eggs. If “germline mosaicism” occurs in a female, her ovaries will contain a mosaic of normal and mutated eggs and thus she will be at substantial risk for having more than one affected male offspring. If the germline mutation occurs in a male, his female offspring are at significant risk for becoming carriers. As a matter of fact, because of the possibility of germline mosaicism, if the mother of an affected male is found not to be a carrier by genetic analysis of her lymphocyte DNA, her risk of having another affected son could be 7–10% ; depending on the degree of mosaicism, in individual cases, this risk could be much higher.

Dystrophinopathies in Females

Almost all patients with dystrophinopathies are male, but rarely females may manifest mild or severe symptoms. 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. 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. 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. Skewed X chromosome inactivation or uneven lyonization has been described in one of monozygotic twins. Twinning could predispose to nonrandom lyonization by reducing the size of the muscle cell population at the time of X chromosome inactivation (i.e. the fewer the cells when lyonization occurs, the greater the chance that the normal X chromosome will be inactivated); alternatively, splitting of the embryonic mass after lyonization could, by chance, result in two embryos with significantly different proportions of active paternal and maternal X chromosomes. Symptomatic females with X-autosomal translocations with breakpoints at Xp21.3-21.2 within the dystrophin gene, resulting in preferential inactivation of the normal chromosome, have also been described. 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. Rarely, uniparental disomy for the entire X chromosome or partial uniparental disomy for the Xp21 region could result in a fully symptomatic female with a DMD or BMD phenotype.

Duchenne Muscular Dystrophy

Clinical Features

In children with DMD, although there is histologic and laboratory evidence (elevated serum CK) of myopathy at birth, clinical manifestations are usually absent during infancy. Short stature, however, may be seen even in presymptomatic children with DMD; at birth, height and weight are normal, but during early childhood, growth curves fall off the normal percentiles for age. During the first two years of life, some affected boys exhibit mildly delayed gross motor development presenting primarily as delayed sitting, standing, and independent walking. The mean age of walking is approximately 18 months (range 12–24 months). Parents of children with DMD usually identify the following first symptoms: gross-motor delay (42%), delay in walking (20%), gait difficulties, including persistent toe-walking and flat feet (30%), learning problems (5%), and speech delay (3%). Many parents also notice frequent falls and calf enlargement. Weakness is usually noted between 2 and 3 years of age; in some cases, however, signs of weakness may be delayed and become apparent after the age of 3 years. Weakness selectively affects proximal limb muscles before distal and the lower extremities before the upper ones, and presents usually as difficulty with running, jumping, standing up from a squatting position, and climbing stairs. The mean age at diagnosis in males with negative family history for DMD is approximately 4 years 10 months, with a range of 16 months to 8 years. Between 3 and 6 years of age, a broad-based waddling gait, exaggerated lumbar lordosis, and calf enlargement are usually observed. Early on, patients 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) ( Figure 30.4A,B ), thus compensating for weak pelvic girdle muscles. Neck flexor weakness often goes unnoticed but it does occur at all stages of the disease; when lying on his back, even at an early stage, a child with DMD is unable to lift his head against gravity. This sign distinguishes boys with DMD from patients with milder phenotypes, such as “Outliers” and BMD, who, at least early on, exhibit preservation of neck flexor strength.

Gowers’ sign in a boy with Duchenne muscular dystrophy. This series of drawings, from left to right, illustrates the various components of a classic Gowers’ maneuver. In the upper panel ( A ), the patient uses hand support on the floor, initially bilateral then unilateral, and hand support on the thighs, either unilateral or bilateral, in order to attain the standing position. The lower panel ( B ) shows the “butt first” maneuver, which evolves into a classic “one-handed” or “two-handed” Gowers’ sign.

Drawings courtesy of Mrs. Julianna Wittrédi, Geneva, Switzerland.

Physical examination shows enlargement of the calf muscles and, in some instances, of the quadriceps, gluteal, deltoid, infraspinatus, and rarely masseter muscles. The calf muscles feel firm and rubbery to palpation. The enlargement, 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) ( Figure 30.5A,B , Figure 30.6 ). With the exception of the sternocleidomastoids, cranial-nerve innervated muscles and also the levator ani and the external sphincter muscles remain essentially unaffected. The external ocular muscles are always spared; the tongue, however, often enlarges. The ankle plantar flexors (gastrocnemius and soleus) and invertors (tibialis posterior) remain very strong until late in the course of the disease while the tibialis anterior and peroneal muscles become weak gradually, resulting in heel cord contractures and toe walking; eventually, this imbalance in muscle strength will lead to bilateral equinovarous foot deformities ( Figure 30.5B ). Neck flexors are weaker than extensors, biceps and triceps are weaker than deltoid, wrist extensors are more involved than flexors, and the quadriceps more than hamstrings.

A boy with Duchenne muscular dystrophy, at the ages of 8 years ( A ) and 11.5 years ( B ). Note enlargement of gastrocnemii muscles bilaterally, known as “pseudohypertrophy.” Also note the progression in foot position from plantigrade ( A ) to mild equinovarus ( B ).

Symptomatic progression of Duchenne muscular dystrophy.

From Jones HR, Burns TM, Aminoff MJ, Pomeroy SL. Nervous System: Part II, Spinal Cord and Peripheral Motor and Sensory Systems. Vol 7 in Netter FH. The Netter Collection of Medical Illustrations, 2 ^nd Ed. Elsevier: 2013, p 265, plate 12-9. All rights reserved.

Between 3 and 6 years of age there may be some evidence of transient improvement, known as the “honeymoon phase” of muscular dystrophy; this is related to the fact that the normal motor developmental progression of the child outpaces disease evolution, but this phase is followed gradually by relentless deterioration. Between age 6 and 11 years, muscle strength decreases almost linearly but the ability to climb stairs, stand from a supine position, climb stairs with rails, go to all fours from supine, and walk a short distance (750 cm) declines rather rapidly in the mentioned order. Loss of function, therefore, does not correlate well with loss of strength of selected muscle groups. When evaluated using the Vignos scale (1, normal function; 9, nonambulatory), most DMD patients remain in functional grade 2 for a long time and then lose function very fast, over a period of 2 or 3 years. The speed at which function is lost, however, may vary substantially among children with DMD. When patients are evaluated with the 6-minute walk test (6MWT), children with DMD younger than 7 years of age may show stability or increase in distance walked (6MWD) over about one year despite progressive muscular impairment. Baseline 6MWD and age (≥7 years) are strong predictors of ≥10% worsening in 6MWD and decline in ambulation in general. In a recent study, baseline 6MWD of <350 meters was predictive of greater functional decline, with loss of ambulation seen only in those patients with a baseline 6MWD <325 meters. Patients ≥7 years of age usually decline, but some may remain stable or even show improvement in their 6MWD over 48 weeks ( Figure 30.7 ). As the disease progresses, deep tendon 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.

( A ) shows change in 6-minute walk distance (6MWD) over 48 weeks in a cohort of 57 placebo-treated DMD patients. ( B ) shows change in percent predicted 6MWD value calculated using age and height. Note the tendency of the 6MWD values to decline in most patients ≥7 years of age and to improve or be stable in patients younger than 7 years of age.

Reproduced with permission from McDonald et al. Courtesy of Allen Reha, MS, PTC Therapeutics, South Plainfield, NJ, USA.

The development of joint contractures is another almost constant clinical feature of DMD. Between age 6 and 10 years, the majority of DMD patients (70%) develop 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. Shoulder contractures occur only late in the disease course. In most patients, the weakness leads to dependency on long leg braces for ambulation at around age 10 years and wheelchair confinement by approximately the age of 11–13 years ( Figure 30.6 ). Weakness of muscles in the upper extremities increases gradually, and wheelchair-dependent patients can perform only minor movements involving their hand and forearm muscles. Muscles of all extremities and trunk become atrophic once ambulation is lost. Wheelchair-bound children tend then to develop scoliosis due to weakness of paraspinal muscles, and gradually their pulmonary function deteriorates related to the weakening of respiratory muscles and probably the increasing scoliosis. The progressive weakness of respiratory muscles is assessed by pulmonary function testing, which shows gradual decline in vital and total lung capacities beginning at the age of 8 or 9 years. With advancing disease, carbon dioxide retention and anoxemia may occur even without infection; nighttime carbon dioxide retention usually presents as morning headaches upon awakening.

The majority of patients die during the period of late teens to late twenties, more frequently from respiratory failure with or without an associated pulmonary infection, but also from cardiac failure secondary to progressive cardiomyopathy. However, survival into the thirties has been observed in patients receiving ventilatory support. In some cases, the immediate cause of death is not apparent. A recent study of patients with molecularly confirmed diagnoses has determined a median survival of 24.0 years, with ventilated patients reaching a median survival of 27.0 years; for unventilated patients, the median survival was 19.0 years. In another study, the overall age for cardiac deaths was 19.6 years (range 13.4–27.5); for respiratory patients without ventilatory support, the age at death was 17.7 years (range 11.6–27.5) but increased to 27.9 years (range 23–38.6) in patients who received mechanical ventilation. In a cohort of patients having both spinal surgery and nocturnal ventilation, the median survival was 30 years.

Becker Muscular Dystrophy

In Becker dystrophy the age of onset of symptoms is usually later, between the ages of 5 and 20 years, sometimes even in the third or fourth decade or later. The mean age of onset is approximately 12 years. Patients with clinical symptoms before the age of 8 years are sometimes difficult to distinguish clinically from DMD patients. By the age of 10 years, almost 50% of the patients are symptomatic and by the age of 20 years almost 90% have clinical signs of the disease. The degree of clinical involvement is milder but the pattern of muscle wasting is similar to the one noted in DMD. Pelvic girdle and thigh muscles are involved first and calf muscle (pseudo)hypertrophy occurs early in most but not all patients. Tibialis anterior and peroneal muscle groups are less affected. Shoulder girdle weakness develops later after the onset of proximal lower extremity weakness. In a series of 67 patients, lower extremity weakness was noted first at the mean age of 11 years, and upper extremity weakness at the mean age of 31 years. Calf muscles, forearm and hand intrinsics remain strong until the late stage of the disease. Calf pain upon or following exercise was reported as a presenting symptom in 28% and as a complaint at some point during the course of the disease in 91% of all patients. Only 2 of the 67 patients presented with myoglobinuria. Thus, muscle pain is very common in patients with BMD but myoglobinuria occurs infrequently. As discussed earlier, in BMD, as in the intermediate type of dystrophinopathy (“Outliers”), there is relative preservation of neck flexor muscle strength; neck flexors become weak later on in the course of the disease. In patients with BMD, facial muscles are usually preserved. Contractures are less likely to develop, and scoliosis is also less common, but it may develop after the patient with BMD is confined to a wheelchair.

Cardiac disease and cognitive impairment are not as common or as severe as in the Duchenne variety, and gastrointestinal symptoms are essentially absent. Rarely, however, cardiac symptoms can be severe in patients with only mild skeletal muscle weakness or can precede muscle weakness by several years. BMD patients with deletions involving the amino-terminal domain are more likely to experience early-onset cardiomyopathy (mid-20s), whereas those with deletions of the rod domain and hinge 3 experience a later onset (mid-40s). Although most patients with BMD have IQs within the normal range, as a group mean IQ is shifted down slightly from that for the general population. Patients with BMD typically remain ambulatory beyond the age of 16 years and into adult life; the mean age of BMD patients when they lose ambulation is in the fourth decade. They usually survive beyond the age of 30 years, with death from respiratory insufficiency or cardiomyopathy/cor pulmonale usually occurring between 30 and 60 years. Cardiac involvement is the cause of death in 50% of patients with BMD. Mean age at death is in the mid-40s. When compared to unaffected brothers, fertility is about 70% in the average BMD patient but below 10% in severely affected individuals.

The mild end of the spectrum includes men with onset of symptoms after the age of 40 years who remain ambulatory even into their 60s. Mildly affected patients with abnormal DMD gene molecular genetic testing and/or dystrophin immunostaining of muscle biopsies have been classified as having (1) “benign” skeletal myopathy with subclinical findings consisting of muscle weakness in the pelvic and/or shoulder girdle with or without associated cardiomyopathy, or (2) BMD with “subclinical” skeletal muscle involvement but with elevated serum CK concentration, calf enlargement, muscle aches and cramps, and myoglobinuria upon exertion.

“Outliers” or Intermediate Phenotype

The intermediate group of patients with mild DMD or severe BMD (called “Outliers”) presents with symptoms intermediate between DMD and BMD. They usually remain ambulatory beyond the age of 13 years but become wheelchair dependent before the age of 16 years. This subgroup of patients was identified during the natural history of DMD study. They can be differentiated from DMD early in life by the preservation of their ability to flex their neck against gravity or against resistance. DMD patients lack this ability even before the age of 5 years, essentially throughout their entire life. Cardiomyopathy and cognitive impairment are also part of the presentation of “Outliers.”

DMD-associated Dilated Cardiomyopathy

In 1987 a five-generation, 63-member family was described who had dilated cardiomyopathy, but no evidence of skeletal myopathy. Towbin et al. showed linkage of DMD-associated dilated cardiomyopathy (DCM) to the dystrophin locus at Xp21 in this family and one other. Subsequent reports defined DMD-associated DCM as a progressive and fatal type of heart disease that presents in the second or third decade of life with congestive heart failure in patients who have little or no skeletal muscle weakness. Males present in their teens and twenties, the disease course is rapidly progressive, and associated ventricular arrhythmias are common. Female carriers develop mild cardiomyopathy in the fourth or fifth decade and exhibit slow progression. The only abnormal biochemical finding is an elevation in serum CK. It has been demonstrated that in patients with the most severe cardiac phenotype, the cardiac muscle is usually unable to produce dystrophin, while skeletal muscle is unaffected.

DMD-associated DCM may be the presenting and main feature in individuals with BMD who have little or no clinical skeletal muscle weakness. Some classify such patients as “subclinical” or “benign” BMD, whereas others may classify such patients as having DMD-associated DCM with increased serum CK activity. In one study of 28 patients with “subclinical” and “benign” BMD ranging in age from 6 to 48 years, 19 (68%) had myocardial involvement, although only two were symptomatic. In another study of 21 patients ranging in age from 3 to 63 years (mean age 40 years), 33% had cardiac failure despite relatively mild skeletal muscle findings.

Different mutations in the dystrophin gene cause selective absence of dystrophin in heart muscle. In several patients with dystrophin-deficient DMD-associated DCM, cardiomyopathy is caused by mutations in the DMD gene that affect the muscle promoter (P _M ) and the first muscle-specific exon (M1) via deletion of P _M and M1 or altered mRNA splicing, resulting in no dystrophin transcripts (M isoform) being produced in cardiac muscle ; however, two alternative promoters that are normally only active in the brain (P _B ) and Purkinje cells (P _P ) are active in the skeletal muscle, resulting in sufficient dystrophin expression to avoid skeletal muscle symptoms. When cardiac dystrophin was tested in two muscle promoter/exon 1-deletion cases, its level was undetectable, whereas skeletal muscle dystrophin levels were normal or reduced to as low as 30%. The exact mechanism for this difference in transcriptional control between cardiac and skeletal muscle remains unclear. A review of all the reported families with DMD-associated DCM points out that mutations involving the 5′ end of the gene result in more severe cardiomyopathy than mutations situated in the spectrin-like region (centered around exons 48–49). With mutations involving the 5′ end of the dystrophin gene, the exclusive cardiac involvement may also be related to a difference in RNA splicing regulation between heart and skeletal muscle. Also, DMD-associated DCM may also be caused by alteration of epitopes in a region of the protein of particular functional importance to cardiac muscle or possibly by mutations in hypothetical cardiac-specific exons. Further, cardiac conduction abnormalities in patients with dystrophinopathies may be related to reduced expression of cardiac sodium channel NA(v)1.5 due to dystrophin deficiency.

The report of a mutation resulting either in late onset cardiomyopathy, or in a Becker phenotype in the same family, raises the possibility of modification of the phenotypic expression of a specific mutation by epigenetic factors.

Muscle Cramps with Myoglobinuria

Exercise-induced myalgias and cramps without skeletal muscle weakness, starting in childhood, have been demonstrated to be caused by mutations in the dystrophin gene of affected members in reported families. In one of the families, nine affected males had high resting serum CK levels, and hypertrophic calves but no clinical evidence of skeletal muscle weakness; all experienced muscle cramps triggered by normal activity, and some reported exercise-induced myalgia and cramps. DNA testing showed a deletion in the first third of the dystrophin gene involving at least exons 10–22. This deletion would be expected to remove the first few repeat units in the central rod-like domain of the protein. Western blot analysis revealed dystrophin of reduced molecular weight but no reduction in the amount of the protein present. One of the authors (BTD) has routinely tested patients presenting with muscle cramps, pains and/or myoglobinuria for dystrophin deficiency (via deletion analysis and in selected cases via muscle biopsy Western blot assay) and has identified only two patients in almost 24 years of neuromuscular practice. One of these two patients, however, had mild to moderate fixed proximal weakness in conjunction with exercise-induced myalgias, cramps, and microscopic myoglobinuria, and carried the diagnosis of BMD. Another family with cardiomyopathy and muscle cramps without clinical weakness proved to have a mutation in the dystrophin gene of the affected members. Although the duration of the follow-up of some of these patients has not been long enough to rule out late-onset skeletal muscular weakness, these cases suggest that dystrophinopathies can sometimes present with signs and symptoms of a metabolic myopathy without permanent or fixed muscle weakness. Myoglobinuria without muscle weakness has also been linked to dystrophin gene mutations in several patients and has been associated with cardiomyopathy and exertional cramps and/or exercise-induced myalgias. As in the previously mentioned cases, a longer follow-up period would be necessary to definitely rule out late-onset skeletal muscle weakness in some of these patients ; it is certain, however, that dystrophin deficiency induced myalgias may persist indefinitely without progression to weakness.

Two molecular regions on the dystrophin gene have been linked to the phenotype of cramps and myalgias: exons 10–44 and exons 45–52, both in the rod-like domain. Some of the patients in these studies have also had associated fixed weakness.

Asymptomatic Elevation of Muscle Enzymes

The incidental finding of an asymptomatic 11-year-old child with elevated CK and a deletion in the region of exons 33–45, together with the essential absence of mutations in the same region in symptomatic patients, has raised the possibility of mutations in the rod domain of the dystrophin gene causing a very mild or even normal phenotype with asymptomatic elevation of muscle enzymes. Also, the “reading frame” theory predicts more BMD than DMD cases assuming the deletion mutations are evenly distributed throughout the gene; nevertheless, the incidence of DMD in the population is much higher than that of BMD. This suggests that many “in-frame” deletions in the rod domain of the dystrophin gene may not be detected because the individuals bearing them are either normal or only minimally symptomatic ; therefore, most probably they will not be seen in neuromuscular clinics or elsewhere, resulting in an underestimation of the prevalence of asymptomatic cases due to an ascertainment bias. It is also possible that some “in-frame” deletions are not detected because they result in non-DMD/non-BMD clinical features.

As discussed before, large “in-frame” deletions of the rod domain may result in severe presentation. A nonsense mutation, truncating the carboxy-terminal domain of dystrophin, has also resulted in an asymptomatic dystrophinopathy in an 8-year-old boy; mRNA analysis failed to implicate an alternative splicing mechanism.

Manifesting DMD/BMD Carrier Females

Carriers are usually free of symptoms but may have mildly increased serum CK and usually mild calf hypertrophy. Two studies have shown that 2.5–7.8% of DMD and BMD carriers exhibit various clinical abnormalities. It should be noted, however, that one study was done before the discovery of the dystrophin gene, and in the other the ascertainment of carrier status was based on pedigree and CK analysis. More recent studies cite an incidence of clinical symptoms between 8% and 22% of female carriers of DMD mutations. Manifesting carriers (MCs) can present with mild but on occasion with severe myopathy of the limb-girdle type or even DMD/BMD. Despite the recruitment of dystrophin-positive satellite cells, severely affected females do not improve over time. If anything, their weakness increases with time due to depletion of satellite cells and thus exhaustion of the regenerative capacity of the muscle similar to that seen in DMD patients. Cardiac involvement is usually subclinical; a cross-sectional study of a Dutch cohort demonstrated that 8% of definite DMD gene mutation carriers had DCM, versus none in definite BMD carriers. Only 38% of the studied population of definite carriers aged 18 to 60 years had a completely normal heart investigation. The remaining had subclinical ECG or echocardiographic abnormalities ( Table 30.3 ). Some carriers, however, can manifest severe cardiac symptoms. Longitudinal follow-up of the same Dutch cohort of 99 DMD/BMD carriers over a period of 7 to 10.6 years identified 11 cases of DCM (10 DMD, 1 BMD); 1 patient died of heart failure at age 57 years. In a different group of 23 proven carriers aged 6.2 to 15.9 years, none had cardiac abnormalities.

Criteria supporting the diagnosis of MCs include a positive family history for DMD or BMD in male patients; heterozygosity for a dystrophin gene mutation; cytogenetic abnormalities (X:autosome translocations with breakpoints at Xp21 being the most common); carrier status proven via linkage analysis; persistently elevated serum CK levels, usually above 1000 IU/L, and myopathic findings in their muscle biopsies in the setting of a positive family history and/or a positive DNA test for a mutation. Thus, a positive family history for DMD or BMD is not always required for the diagnosis of MCs (“isolated” MCs). In a large study of girls and women with neuromuscular complaints, about 10% of the isolated cases of hyperCKemic myopathy were found by dystrophin analysis to have a dystrophinopathy as the cause of their disease. Their muscle biopsies showed a mosaic of dystrophin-positive and dystrophin-negative fibers ( Figure 30.8E, F ). Females with a disease-causing DMD gene mutation may be symptomatic also if they have Turner syndrome (i.e. complete or partial absence of an X chromosome), X chromosome uniparental disomy, male pseudohermaphroditism due to a mutation in the androgen receptor gene, or nonrandom X chromosome inactivation. The inactivation status of a female’s X chromosomes can be measured in leukocytes or muscle tissue; however, some studies have shown no consistent correlation between X chromosome inactivation (XCI) ratios in leukocytes and serum CK activity, severity of clinical manifestations, or the proportion of dystrophin-negative fibers detected in muscle biopsies. In another study of 7 proven MCs, the XCI pattern was skewed toward nonrandom (greater than 80:20) in the 4 out of 7 with deletions or duplications but was random in all 3 with nonsense mutations. In contrast, Pegoraro et al. showed that more than 90% of heterozygous females with skewed XCI (defined as equal or greater than 75:25) in leukocytes developed mild, moderate, or severe symptoms. The occurrence of both highly skewed and completely random XCI patterns in MCs suggests lack of clear correlation possibly related to: (i) different patterns of XCI in lymphocyte DNA and muscle tissue in certain patients, or (ii) difference in methylation status between the DMD locus and the androgen receptor gene locus, which is usually tested in methylation assays.

( A , B ) Immunostaining of frozen sections of human skeletal muscle biopsies from unaffected controls and Duchenne muscular dystrophy (DMD) patients with antidystrophin antibodies using indirect immunofluorescence. A also displays hematoxylin and eosin-stained sections corresponding to unaffected and DMD biopsies, respectively. There is complete absence of immunofluorescence at the sarcolemma in a muscle section from a DMD patient, compared with the homogeneous staining of the plasma membrane in normal, unaffected muscle ( A , B ). In B , muscle biopsies have been immunostained with a combination of antidystrophin (DYS2) and ankyrin 1 (ANK1) antibodies; the latter stain the sarcomere green. ( C ) Immunofluorescence of frozen section of muscle tissue from a Becker muscular dystrophy (BMD) patient immunostained with antidystrophin antibodies. Note the discontinuous and weak labeling in all muscle fibers in BMD. ( D ) Immunostaining of muscle biopsy from normal female control. ( E , F ) Immunostaining of muscle biopsies from symptomatic females with antidystrophin antibodies. Note the mosaic pattern with complete loss of immunofluorescence in certain fibers (yellow, red arrows) and partial loss in others (blue arrows).

A and B courtesy of Matthew Alexander, PhD, Division of Genetics, Boston Children’s Hospital. A has been reprinted from Molecular Genetics and Metabolism , Vol 74, O’Brien KF and Kunkel LM, Dystrophin and muscular dystrophy: past, present, and future, pages 75–88, Copyright © 2001, with permission from Elsevier. C has been reproduced from Muntoni et al., with permission from BMJ Publishing Group, Ltd. D, E, and F courtesy of Dr. Hart Lidov, Department of Pathology, Boston Children’s Hospital.

Monozygotic female twins with DMD gene mutations typically show discordance in manifesting the disease due to skewed X chromosome inactivation in the manifesting twin (discussed earlier in the subsection “Dystrophinopathies in Females”). Mutations in both DMD alleles in MCs have also been described.

Limb-girdle Muscular Dystrophies

In childhood, the clinical presentation of certain limb-girdle muscular dystrophies (LGMDs) resembles DMD or BMD except for the absence of cognitive deficits, the less frequent occurrence of cardiomyopathy, and the less marked calf hypertrophy. LGMDs comprise a wide clinical spectrum associated with elevated levels of serum CK and thus, at times, are difficult to distinguish from a sporadic case of DMD or BMD. LGMDs are transmitted in an autosomal recessive or dominant fashion; thus, if there is a clear evidence for autosomal transmission, a dystrophinopathy can easily be excluded. If not, negative genetic testing for a DMD gene mutation will exclude a dystrophinopathy and raise the possibility of an LGMD. LGMD2I is the type of autosomal recessive LGMD that closely resembles DMD/BMD because of the frequent respiratory and cardiac involvement; it is caused by mutation in FKRP , the gene encoding fukutin-related protein. Details on the diagnostic approach to LGMDs can be found in Chapter 2 , “Genetics of Neuromuscular Disorders,” and also in Chapter 34 on LGMDs.

If symptoms of limb-girdle dystrophy are noted in a girl, one should always consider the rare possibility of a manifesting DMD or BMD carrier.

Acid Maltase Deficiency, Spinal Muscular Atrophy

Acid maltase deficiency (AMD) and spinal muscular atrophy (SMA) may both present in childhood, before the age of 5 years, with proximal muscle weakness and sometimes calf muscle hypertrophy; however, the serum level of CK (usually less than 1000 IU/L in SMA, an increase in AMD smaller than the increase in DMD), the muscle biopsy findings (i.e. glycogen storage in AMD), and electromyography will easily permit the distinction from DMD. SMA is caused by mutations in the SMN gene (chromosomal locus 5q11-q13); molecular genetic testing is easily available to confirm the diagnosis of SMA.

Emery-Dreifuss Muscular Dystrophy

Emery-Dreifuss muscular dystrophy is another X-linked or autosomal dominant or recessive muscular dystrophy that can at times be confused with BMD at the onset of the symptoms. However, the lack of pseudohypertrophy, the mild CK elevation, the selective involvement of biceps and triceps muscles, and the early occurrence of elbow and heel contractures will allow one to differentiate them clinically; genetic testing for Emery-Dreifuss muscular dystrophy is available (emerin, lamin A/C, FHL1 and other gene mutation analysis) to confirm the diagnosis.

Congenital Muscular Dystrophies

As mentioned previously, in very rare instances dystrophinopathies can present similarly to congenital muscular dystrophies with neonatal weakness, hypotonia, and subsequent developmental delay. The differential diagnosis can be made clinically by the early onset, the modestly increased CK level (albeit maybe markedly elevated in congenital muscular dystrophies also), and the frequent facial involvement and contractures seen in congenital muscular dystrophies. Genetic testing and muscle biopsy immunostaining may also be helpful in differentiating the two entities.

Mitochondrial DNA Depletion Syndrome

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive mitochondrial cytopathy presenting with a wide spectrum of clinical manifestations, such as weakness, hypotonia, developmental delay, and/or seizures, and categorized as myopathic, encephalomyopathic, hepatocerebral, or multisystemic forms. Patients with MDS may have involvement of single or multiple organs, including the central and/or peripheral nervous system, muscle, liver, and kidney. Vu et al. described five patients with MDS; age at onset ranged from 3 months to 5 years, and two patients had laboratory and clinical features suggestive of dystrophinopathy. CK levels in these two patients were high (4229/6000 IU/L) and muscle biopsies in all five showed abundant ragged-red fibers. Thus, mtDNA depletion should be considered in children with weakness and high CK values, particularly if associated with multiorgan involvement (e.g. liver, brain, peripheral nerves).

Dermatomyositis

Dermatomyositis before the appearance of the skin rash can be mistaken for a dystrophinopathy, but the acute or subacute onset of symptoms and the muscle MRI and/or biopsy findings usually permit easy distinction from DMD or BMD.

Elevation of Liver Enzymes

The liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are also present in muscle and thus are elevated in DMD and BMD. Therefore, an unexplained persistent elevation of liver enzymes should always alert the clinician to the possibility of an underlying muscular dystrophy; this is true even in females, who could prove to be symptomatic carriers, asymptomatic carriers of DMD/BMD, rarely symptomatic patients with DMD/BMD (e.g. if they have Turner syndrome), or patients with other muscular dystrophies. 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. See Case Example 30.1 .

Case Example 30.1

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