Muscular Dystrophies and Allied Disorders I: Duchenne and Becker Muscular Dystrophy





Background


Although the advent of molecular genetics has transformed the diagnostic confirmation of Duchenne and Becker muscular dystrophy, we thought it worth retaining some historical background and clinical description of these classical muscular disorders.


Duchenne muscular dystrophy (DMD) has been recognized as a clinical entity since the 19th century. In 1868, Duchenne gave a detailed account of 13 male patients with progressive muscle weakness ( ). Onset was in childhood, and weakness was initially worse in the lower limbs and lumbar region and was accompanied by hypertrophy of some muscles. He also took muscle samples from these cases using a biopsy needle that was the forerunner of the type we use today (see Ch. 1 ) and noted the marked proliferation of connective tissue and adipose tissue. Although the disease is attributed to Duchenne, earlier descriptions had in fact been made by the London physician Meryon and by Conte and Gioja in Italy ( ). Meryon’s life and work have been researched and published ( ), and Conte’s contribution has been celebrated by Nigro with the establishment of a Conte Foundation (1986). Meryon described the pathological features of autopsy samples from his cases and pointed out the necrotic nature of the muscle but apparent normality of the nervous system. He also suggested that the sarcolemma was principally at fault and that genetic transmission was through females and only affected males.


In 1879, Gowers presented clinical observations on 21 of his own cases and reviewed the descriptions of over 100 cases of others ( ). He observed and illustrated the unusual way affected boys get up off the floor by climbing up their legs – the Gowers’ manoeuvre. Despite the advances in clinical management over the years, Gowers’ masterly description of the devastating effects of the disease is still very apt (see ).


In 1955, Becker and Kiener described cases with similar features to Duchenne dystrophy but milder in severity ( ). There have been several subsequent reports on similar cases of Becker muscular dystrophy (BMD), and in 1984 it was shown by linkage analysis to be allelic to DMD on the short arm of the X chromosome ( ).


DMD is one of the most common neuromuscular disorders, with an incidence that is often quoted as approximately 1 in 3500 live male births, but more recent estimates are 8.9 live male births to 15.9–19.5 per 100,000 live births with some geographical variation ( ). The gene is highly prone to mutations and is a common childhood disorder. The Becker form is less common, with a predicted incidence of 7.29 per 100,000 live male births ( ), although within the general population as a whole their prevalence is similar, as BMD patients live longer ( ).


The second edition of this book, published in 1985, was written before the explosion in molecular science, and the underlying cause of the disease was not known. At that time, all that was known was that the locus for DMD was on the short arm of the X chromosome at Xp21 and that the Becker form was probably allelic. Subsequently, the gene responsible for both has been cloned and its protein product named dystrophin ( ). Considerable knowledge of the consequences and types of mutations in the gene, and the relationship of dystrophin to other proteins, has been acquired, but the precise function of dystrophin is still being investigated. Clinical management has greatly improved over the years ( ), and various therapeutic strategies are being investigated (see below), but there is still no cure and the relentless progression of the disease in Duchenne patients inevitably leads to premature death, although more affected patients now survive into adulthood. The gene for dystrophin was the first to be identified in a neuromuscular disorder and there are several texts documenting its cloning and structure (see ). Muscle biopsies are an important component of the assessment of a patient with a muscular dystrophy, and immunohistochemistry is now an essential component of differential diagnosis. Muscle biopsies are now performed less often on possible Duchenne/Becker patients and there is a tendency to rely on the results of molecular analysis. The distinction between DMD and BMD, however, is a clinical one, although there is often a correlation with the amount of dystrophin expressed, which requires a muscle biopsy. The frame-shift hypothesis ( ) does not explain the amount of dystrophin produced in association with all mutations, and secondary pathological changes can be informative. In addition, muscle pathology and the amount of dystrophin induced is an important outcome measure in clinical trials (see below).


Clinical Features


The clinical manifestations of DMD and BMD are now well known and documented (see ). The aim of this summary ( Table 10.1 ) is to draw attention to features that should alert pathologists and that they may find helpful in arriving at a diagnosis. Neonatal screening of serum creatine kinase (CK) activity can detect affected cases at birth, but clinical abnormalities are not usually apparent until the child starts to walk. Motor milestones such as sitting, standing and walking are often delayed, and about 50% of cases of DMD are late walking (beyond 18 months of age). Contractures of the Achilles tendons and hip flexors are early features of DMD and result in toe-walking. Affected patients have a characteristic waddling gait and lumbar lordosis. Scoliosis occurs after loss of ambulation and may require surgical intervention. Duchenne and Becker boys are prone to falls and get up with the typical Gowers’ manoeuvre. Duchenne boys are usually unable to jump or hop, but these may be achieved in a Becker patient. Muscle weakness is proximal more than distal, is greater in the legs than the arms and is steadily progressive. Hypertrophy of calf muscles is an early feature but is not specific to DMD and BMD. This is a pseudohypert-rophy (a term coined by Duchenne), resulting from an increase in fibrotic and adipose tissue. Cramps on exercise may also be a feature in BMD patients more than DMD. In some mild cases of BMD this may be the presenting feature. Rhabdomyolysis may also occur and, although a particular feature of some metabolic and other disorders, this should not mislead the pathologist (see Ch. 20 , Table 20.2 ). There may be a delay in learning to speak and a variable degree of non-progressive intellectual impairment in about 30% of DMD patients, but this is rare in cases of BMD.



TABLE 10.1

Main Features of Duchenne and Becker Muscular Dystrophy

























































































Duchenne Muscular Dystrophy Becker Muscular Dystrophy
Clinical Features
Manifests within the first 5 years Manifests at any age (childhood-adulthood)
Delayed motor milestones Motor milestones may be delayed, often normal
Progressive proximal weakness Variable weakness (asymptomatic/mild/severe)
Contracture of Achilles tendon resulting in toe-walking Toe-walking variable
Waddling gait, lumbar lordosis Waddling gait, lumbar lordosis variable
Difficulty hopping, jumping, never run Can usually run, motor difficulties variable
Difficulty rising from floor (Gowers’ manoeuvre) Motor difficulties variable
Difficulty going up stairs
Calf hypertrophy Calf hypertrophy
Cramps rare Cramps common
Rhabdomyolysis rare Rhabdomyolysis common
Ambulation
Lost by 12 years (without steroid therapy) Usually ambulant into adolescence or adult life
Creatine kinase
Usually grossly elevated (10–50 times normal) Usually grossly elevated even in mild cases
Elevated at birth, often grossly May be elevated at birth
Associated Features
Cardiomyopathy invariable by late teens Cardiomyopathy variable but common
Intellectual impairment common; severe in about 30% Intellectual impairment rare
Nocturnal hypoventilation Nocturnal hypoventilation rare
Scoliosis common after loss of ambulation Scoliosis rare if ambulant
Respiratory failure by late teens Respiratory failure rare
Pathology
Clinical severity cannot be assessed from pathology Clinical severity cannot be assessed from pathology
Necrosis, regeneration, fibrosis, wide variation in fibre size, variable adipose tissue Necrosis, regeneration, fibrosis, wide variation in fibre size, variable adipose tissue
Dystrophin usually absent except on revertant fibres Dystrophin usually present but amount reduced
Reduction of all dystrophin-associated proteins Reduction of all dystrophin-associated proteins
Pronounced up-regulation of utrophin on mature fibres Utrophin often up-regulated on mature fibres


In the past, most Duchenne boys died before the age of 20 years, but better management, in particular non-invasive respiratory support at the onset of respiratory failure, has considerably improved life expectancy, with survival even into the 30s. Cardiac involvement is present in most cases of DMD by the late teens and is common in BMD, but improved therapeutic and management strategies have improved outcomes ( ). Steroid therapy is now widely used from an early age and has proved to be beneficial for motor function. The clinical manifestations of the disease established prior to therapeutic intervention have to some extent now been altered.


Several serum enzymes are elevated in DM. Estimation of CK is the most sensitive and reliable, and levels grossly elevated. In both DMD and BMD, levels are usually very high, 50–100× the normal level, and they are particularly elevated early in the disease and may drop during its course. Levels of < 10 times the normal are more likely to be associated with other forms of muscular dystrophy. Most cases have a raised CK, but some cases with a mutation that are relatively asymptomatic can have a normal CK. There is no reported case of DMD with normal CK. The level of CK does not correlate with clinical severity, and equally high levels occur in both DMD and BMD.


Clinical severity is variable in both DMD and BMD and may even vary in members of the same family who carry the same mutation. The distinction between DMD and BMD is a clinical one, and there is a spectrum of severity, in particular in the degree of weakness and the age at which ambulation is lost. Classical Duchenne patients lose ambulation at a mean age of about 9 years and are almost invariably off their feet by the age of 12 years, although steroid therapy can delay loss of ambulation. Becker patients, however, are generally ambulant to 16 years or beyond. The severity of BMD is very wide, and several cases have been documented in which the patients have minimal muscle weakness and lead near-normal lives for decades. At the severe end of the Becker spectrum there are patients approaching the Duchenne type in severity but who remain ambulant after 12 years of age and go off their feet before 16 years of age, or later. These so-called ‘intermediate’ cases often have atypical mutations, particularly in the 5′ region of the dystrophin gene, and they highlight the value of muscle biopsy and of examining protein expression.


Histology and Histochemistry


The classical histological changes in DMD and BMD are rounding of the fibres; diffuse variation in fibre size, with hypertrophy and atrophy of both fibre types; necrosis and subsequent loss of fibres; basophilic regenerating fibres; densely stained hypercontracted fibres; increased internal nuclei; proliferation of endomysial and perimysial connective tissue; increased adipose tissue; and sometimes an increased cellular response ( Fig. 10.1 ).




Fig. 10.1


Sections stained with H&E from (a and b) two cases of Duchenne muscular dystrophy aged 9 and 6 years, respectively, and (c and d) two cases of Becker muscular dystrophy aged 4 and 6 years, respectively, showing typical dystrophic features. (a) There is a wide variation in fibre size (range 10–90 μm); the fibres are round in shape and have excess connective tissue round most of them. A few internal nuclei (small arrow), a darkly stained hypercontracted fibre (∗) and a little adipose tissue (large arrow) are also shown. (b) There are several pale necrotic fibres (o), a few basophilic fibres (green arrow) and several hypercontracted fibres (∗; up to 80 μm in diameter). (c) From the 4-year-old Becker patient, the same features are present but are less pronounced (fibre size range 15–60 μm). (d) The necrotic fibres are invaded by macrophages ( ) (fibre size range 30–95 μm).


These features collectively are often referred to as ‘dystrophic’, and they reflect the progressive loss of muscle and necrotic nature of the tissue. Care should be exercised in describing muscle as ‘dystrophic’ when only excess connective tissue is present, as this can occur in the absence of necrosis. Clinical severity cannot be judged from the degree of pathology; it is not possible to distinguish Duchenne and Becker dystrophy on the basis of histology alone (see Fig. 10.1a, d ).


Pathological changes can be seen in DMD, even at a few months of age, when there are no clinical manifestations of the disease other than elevated CK ( Fig. 10.2 ). As the disease progresses, regeneration fails to keep pace with necrosis and the muscle is gradually replaced by connective and adipose tissue ( Fig. 10.3 ). Abnormalities in BMD patients as young as 1 year can also be seen.




Fig. 10.2


Muscle biopsy from a 6-month-old preclinical case of Duchenne muscular dystrophy. Note the abnormal features even at this stage, in particular variation in fibre size, increased endomysial connective tissue, adipose tissue and internal nuclei (arrow; H&E).



Fig. 10.3


Two sections of advanced muscular dystrophy from (a) an 18-year-old case of Duchenne muscular dystrophy and (b) a 28-year-old case of Becker muscular dystrophy who were both re-biopsied to assess their dystrophin status. (a) Extensive adipose tissue is shown with some residual muscle fibres; (b) shows extensive fibrotic material containing fragments of fibres haematoxylin and eosin (H&E).


Attempts have been made, prior to the molecular era, to identify pathological changes in the muscle of potentially dystrophic male fetuses but these are equivocal and difficult to assess ( ). Immunolabelling of dystrophin in at-risk fetuses, however, can be useful, but with improvements in molecular techniques this is now rarely performed ( ).


Changes in Fibre Size


All cases show variation in fibre size, which is often marked. Fibre morphometry is rarely necessary when assessing cases of DMD as the size variation is usually obvious. In some mild BMD cases, however, variation may be less apparent but is usually present. The smallest fibres (< 5 μm) may be barely visible with routine histological stains but may be more apparent with immunolabelling (see below). Some hypertrophic fibres ( Fig. 10.4 ) can be extremely large (> 200 μm) and almost give the impression of several fibres joined together. Branching of fibres occurs and contributes to the variation in fibre size (see Fig. 10.1a ).




Fig. 10.4


A very hypertrophic fibre 250 μm in diameter that shows splits and multiple internal nuclei, some of which are associated with the splits (arrow). Note also the adjacent split and whorled fibre (H&E).


Abnormalities in Distribution


The size variation is diffuse, with no groups of large fibres. Occasionally, clusters of small fibres may be seen and this has sometimes been put forward as evidence of denervation ( Fig. 10.5 ). Many of these fibres express fetal myosin, so may be regenerating, but this alone does not distinguish them from non-innervated or denervated fibres (see Ch. 6 ). The overall picture, however, is myopathic and there are never any signs of reinnervation or fibre type grouping.




Fig. 10.5


Clusters of small fibres in a case of Becker muscular dystrophy that are sometimes mistaken for denervation. Note also the darkly stained hypercontracted fibres (∗) and the excess fibrous tissue (H&E).


Fibre Typing


Fibre type differentiation is usually impaired. With oxidative enzymes, it is often indistinct (particularly in DMD, and less so in milder/BMD cases), and most fibres show an intensity intermediate between normal type 1 and type 2 fibres. Fibres devoid of cytochrome c oxidase are rare but can occur. Fibre typing with ATPase is also not always clear, particularly at pH 9.4, although it may be visible with acid preincubation. This poor differentiation is probably due to the presence of more than one myosin isoform in several fibres (see below). When fibre typing is visible, type 1 fibres (slow fibres) often predominate, a common myopathic feature ( Fig. 10.6 ); 2C fibres are also common, some of which may express more than one isoform of myosin, and the basophilic regenerating fibres stain as 2C fibres. The variation in fibre size affects both main fibre types (see Fig. 10.6 ), although the largest fibres in young patients may more frequently be type 2.




Fig. 10.6


Biopsy of a case of Duchenne muscular dystrophy stained for ATPase at pH 9.4 showing variation in size of both fibre types and a marked predominance of the pale type 1 fibres. Several of the dark fibres are 2C fibres in this section and were also dark when stained for ATPase with acid preincubation at pH 4.6 and 4.3. Fibre typing at pH9.4 is often not as clear as this


Changes in Sarcolemmal Nuclei


Internal nuclei displaced from their normal sarcolemmal position are common, but the number of affected fibres is not as great as in some other muscular dystrophies (see Fig. 10.1a ). Multiple internal nuclei within one fibre in cross-section are rare in children with DMD but may occur in BMD and are common in other forms of muscular dystrophy: exceptions are the large fibres with splits in which there may be multiple internal nuclei, many of which are associated with the splits (see Fig. 10.4 ). Regenerating, basophilic fibres may have large internal vesicular nuclei with a prominent nucleolus and pale nucleoplasm ( Fig. 10.7 ).




Fig. 10.7


A cluster of blue basophilic regenerating fibres with large nuclei (size range 15–20 μm) in a case of Becker muscular dystrophy (H&E).


Degeneration and Regeneration


Necrotic fibres may be isolated or clustered, and phagocytes are often seen within them (see Fig. 10.1d ). The necrosis is often segmental and only affects a portion of the fibre. Necrotic fibres appear pale with haematoxylin and eosin (H&E) staining and with the Gomori trichrome technique (see Fig. 10.1b, d ) but may retain their differential myosin content. The large, round hypercontracted fibres that are intensely stained with histological techniques are damaged fibres (see Fig. 10.1a ; Fig. 10.8 ). There is still controversy as to their significance, but they are a particular feature of DMD and BMD and sometimes more numerous and more easily seen with the Gomori trichrome stain. Fibres that lack glycogen are also common in DMD, but they are not a specific feature and probably represent a stage in muscle damage, prior to necrosis. Regenerating, basophilic fibres may also occur in clusters or be diffuse throughout the sample (see Fig. 10.7 ). A variable number of fibres that are not basophilic show developmental myosins and may be at various stages of maturation (see below).




Fig. 10.8


Rounded fibres (size range 25–85 μm), most of which are surrounded by excess connective tissue and fat in a case of Duchenne muscular dystrophy. Note also the dark hypercontracted fibre (∗) and a few slightly basophilic fibres (arrow; H&E; compare with Fig. 10.1b ).


Fibrosis


Both perimysial and endomysial fibrosis are variable in extent and are a consistent feature of DMD and BMD. In early cases it may only be slight but it is often pronounced, and often all fibres are surrounded by excess endomysial connective tissue (see Fig. 10.8 ). Excess adipose tissue is often perimysial, but it can also occur in the endomysium.


Cellular Reactions


A variable number of cells of different type may be apparent in a Duchenne or Becker sample. Areas of necrosis frequently appear cellular with a mixture of macrophages, T cells and myoblasts, and mast cells are also common. Eosinophils and B cells are rarely seen.


Architectural Changes


Whorling of the myofibrils is common, particularly in the hypertrophic fibres ( Fig. 10.9a ). These are seen well with oxidative enzyme stains such as reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR). Moth-eaten fibres ( Fig. 10.9b ) and varying degrees of disruption of the myofibrils may also be apparent, although less so than in other dystrophies, and some aggregation of oxidative enzyme stains may be seen (see Fig. 10.9a ). Core-like areas devoid of mitochondria are infrequent in DMD and BMD but can occur.




Fig. 10.9


Various architectural changes revealed with staining for NADH-TR. Note in (a) variable degrees of whorling of myofibrils (large arrows) and aggregation of stain (small arrow; fibre size range 10–60 μm), and in (b) some moth-eaten fibres (28–32 μm) with areas that lack stain (arrow). Fibre typing is clear in (a) but less distinct in (b).


Immunohistochemistry


Dystrophin


Cloning of the gene responsible for DMD and BMD and localization of its protein product, dystrophin, to the sarcolemma led the way to the revolution in muscle pathology that has occurred during recent decades (see ). The gene for dystrophin is one of the largest known, with 2.5 Mb of DNA and 79 exons. It has some very large introns, and the transcribed mRNA is 14 kb. Transcription of the gene is thought to take about 16 hours. The full-length protein has a predicted molecular mass of 427 kDa and has four main domains ( Fig. 10.10 ); the commercial monoclonal antibodies commonly used recognize epitopes in these different domains (see Table 6.1 and Fig. 10.10 ). The N-terminus is an actin-binding domain; the large rod domain has 24 spectrin-like repeats and four hinge regions, and recent studies have revealed the complex nature of this region and indicated that the concept of a simple rod shape of this domain needs to be revisited ( ); the cysteine-rich domain binds β-dystroglycan; and the C-terminal domain binds syntrophin, dystrobrevin and probably also F-actin and α-actinin. The dystrophin gene has at least eight promoters that give rise to different isoforms of different molecular mass ( Fig. 10.11 ), and there is considerable splicing at the 3′-end. In addition, internal ribosome entry sites, for example in exon 5 – have been identified ( ). The various isoforms of dystrophin are differentially expressed in skeletal, cardiac and smooth muscle, fetal muscle and neural tissue. It has been suggested that the mental retardation that occurs in about 30% of DMD cases may relate to mutations in the C-terminal domain and involvement of the brain isoforms ( ). In skeletal and cardiac muscle the full-length transcripts from 5′ promoters are the most important, and antibodies corresponding to the different domains of the protein show uniform labelling of the sarcolemma of all fibres in normal muscle, and in disorders not caused by defects in the DMD gene. The various isoforms of dystrophin share a common C-terminus (see Fig. 10.11 ), and antibodies with epitopes in this region (e.g. DYS2 from Leica) recognize all isoforms. On full-length immunoblots using antibodies to the N-terminus or rod domain (e.g. DYS1), dystrophin appears as a doublet, probably representing the 427 and 400 kDa muscle isoforms. With antibodies to the C-terminus (e.g. DYS2), dystrophin is seen as a single band on immunoblots (DYS3 does not recognize denatured dystrophin on immunoblots). Several other antibodies to dystrophin are also commercially available (see Ch. 6 ).


Feb 23, 2021 | Posted by in MUSCULOSKELETAL MEDICINE | Comments Off on Muscular Dystrophies and Allied Disorders I: Duchenne and Becker Muscular Dystrophy
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