Duchenne Muscular

Dystrophy

Introductory article
Article Contents
. Introduction

Marek Michalak, University of Alberta, Edmonton, Alberta, Canada

Michal Opas, University of Toronto, Toronto, Ontario, Canada

. The Dystrophin Gene

. Dystrophin
. DystrophinGlycoprotein Complex

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects muscles
and causes progressive weakness. Dystrophin, the product of the DMD gene, is one of
several membrane proteins that form the dystrophinglycoprotein complex, which helps
to maintain the integrity of muscle cells; loss of these proteins leads to the wasting of
muscle cells and to the pathology of DMD.

Introduction
Duchenne muscular dystrophy (DMD) is one of the most
frequent X-chromosome-linked recessive disorders, occurring in 1 in every 3500 male births. The gene mutated in
DMD encodes for the protein dystrophin. DMD has a very
high mutation rate with characteristic and severe clinical
features. In DMD, a progressive proximal muscle weakness becomes noticeable during early childhood. Muscle
strength continually diminishes and leads to wheelchair
dependence by approximately 12 years of age. Death
usually occurs from chronic respiratory insuciency and/
or cardiac failure. Histologically, DMD is characterized by
progressive degeneration of muscle bres. Although
skeletal muscle weakness is the predominant symptom,
progressive cardiomyopathy is common and can be severe.
In addition to muscle pathology, intellectual impairment
of varying degrees is present in about 30% of all patients
with DMD. Cardiomyopathy is also a predominant
clinical problem in Becker muscular dystrophy (BMD), a
milder variant of DMD. BMD is less common than DMD
and has an estimated incidence of approximately 1 in
18 500 births. Clinical manifestation includes slowly
progressive weakness of proximal limb girdle muscles,
hypertrophy of calf muscles and high levels of serum
creatine kinase in addition to cardiomyopathy. Patients
with BMD are usually able to walk and have a normal
lifespan.

The Dystrophin Gene

The gene responsible for DMD and BMD was identied in
1987; it produces a protein called dystrophin. The DMD
gene is one of the most complex genes identied to date,
with a length of over 2400 kilobases (kb) encoding 79 exons
at XP21.1 of the human X-chromosome. This is the largest
gene so far identied in humans, covering more than 2.5
megabases. The gene contains up to eight alternative

. Function of Dystrophin
. Therapies
. Conclusions

promoters. Three promoters located in the 5 region of the

dystrophin gene give rise to the full-length transcription of
a 14-kb messenger ribonucleic acid (mRNA). The 14-kb
mRNA encodes for dystrophin, a protein with a molecular
weight of more than 420 kDa, which is expressed predominantly in skeletal, cardiac and smooth muscle and, at
lower levels, in brain. Each of the three promoters is linked
to a unique rst exon for transcription of each of the three
dierent isoforms in a tissue-specic manner. Three
promoters located at the 3 end of the dystrophin gene
control the expression of tissue-specic, alternative DMD
gene products including proteins expressed in the central
nervous system, in Schwann cells of the peripheral nerves,
and in many tissues such as smooth muscle, embryonic
skeletal muscle and cardiac muscle. Alternatively spliced
isoforms of the C-terminal region of dystrophin have been
identied in many other tissues.
In addition to this array of dystrophin isoforms, a gene
on chromosome 6 encodes a homologue of dystrophin,
named utrophin. Utrophin has structural and amino acid
sequence similarities to dystrophin. It associates with the
plasma membrane and is also found in low levels in DMD.
Utrophin is enriched at the neuromuscular junction where
it may play a role in synaptic formation. A new dystrophinrelated protein (designated DRP2) was recently identied.
DRP2 is a 110-kDa protein similar to the dystrophin. A
gene localized at Xq22 encodes the protein and is expressed
mainly in the brain and spinal cord. DRP2 may be a
member of a new family of dystrophin-like proteins.

Dystrophin
Dystrophin normally resides in skeletal, cardiac and
smooth muscles and is also present in much lower amounts
in the brain and peripheral nerves of several species. In
both muscles and neurons, dystrophin is found in the
plasma membrane and is enriched at the myotendinous
junction and at the postsynaptic membrane of the

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Duchenne Muscular Dystrophy

neuromuscular junctions. Dystrophin is a cytoskeletal

protein that binds to actin and interacts with a dystrophin
glycoprotein complex (DGC) which bridges the cytoskeleton, the basal lamina and the plasma membrane. The
DGC is made up of integral and peripheral membrane
proteins organized into three distinct entities: dystroglycan, sarcoglycan and syntrophins (Figure 1).
In DMD, dystrophin is absent from skeletal and cardiac
muscles as revealed by both immunocytochemical and
immunological analysis of skeletal muscle biopsy. In 80%
of patients with BMD, dystrophin is expressed in truncated
forms and is detected as a protein of smaller size by
immunological methods. The dystrophin gene in over half
of patients with DMD and BMD has partial deletions, with
the majority of deletions occurring in 2-kb region in the
middle. Deletion size has no direct correlation to the
clinical severity of the disease. On the other hand, in BMD
dystrophin gene deletions maintain the open reading frame
for amino acids and are predicted to make shorter proteins
of lower molecular weight with internal deletions. These
smaller dystrophin gene products are presumably semifunctional and result in milder clinical phenotypes.
The muscle form of dystrophin contains 3685 amino
acids organized in four distinct structural domains
(Figure 1). The N-terminus of the protein, which binds Factin, exhibits signicant amino acid sequence homology
to a-actinin. It is followed by a long, rod-like, central
domain comprised of 24 triple helical coiled-coil repeats

similar to those found in spectrin. New F-actin-binding

sites are also found in the middle of the dystrophin rod
domain. The C-terminal domain of dystrophin may have
additional site(s) for F-actin attachment. Thus, dystrophin
may bind alongside an actin lament via multiple binding
sites (Figure 1). Dystrophin, therefore, is probably an
elongated rod-shape, like spectrin, and might also be
dimerized in an antiparallel fashion. This region of the
protein also contains some proline-rich sequences, which
may lead to a breakdown in its repetitive spectrin-like
organization. It has been postulated that these proline-rich
hinge sequences may confer exibility on the central rodlike structure.
The C-terminal region of dystrophin can be divided into
two separate domains. There is a cysteine-rich domain that
shows some amino acid sequence similarity to the slime
mould a-actinin. This is followed by the C-terminal 325
amino acids. The cysteine-rich domain interacts with the
cytoplasmic tail of the b-dystroglycan and binds syntrophins (Figure 1). It contains an EF-hand-like calciumbinding consensus amino acid sequence, a zinc nger-like
(ZZ) motif. This region of the protein binds calcium and
zinc. A tryptophan-enriched (WW) domain is also found in
the cysteine-rich region of dystrophin. The WW domain
binds proline-rich peptide motifs which may mediate
interactions with the b-dystroglycan bound syntrophins
(Figure 1). It is well documented that quantities of the DGC
are drastically reduced in patients with DMD in whom
-Dystroglycan

Muscle plasma membrane

-Dystroglycan
Sarcoglycans

Dystrophin
NH+3

COO
Actin filaments

Homology
to -actinin

Spectrin-like
domain

Cysteine-rich
domain

C-terminal domain

Syntrophins

Key
Carbohydrate

Figure 1 Dystrophinglycoprotein complex (DGC). DGC is composed of dystrophin, dystroglycan (a-dystroglycan (156 kDa), b-dystroglycan
(43 kDa)), sarcoglycans (a 50-kDa a-sarcoglycan, a 43-kDa b-sarcoglycan, a 35-kDa g-sarcoglycan, a 35-kDa d-sarcoglycan) and syntrophins (a 54kDa a-syntrophin, a 58-kDa b-syntrophin). a-Dystroglycan binds the extracellular matrix proteins, laminin and agrin, and may interact with sarcoglycans.
The cytoplasmic tail of b-dystroglycan binds to the C-terminus of dystrophin and anchors the protein to the DGC. F-actin (actin filaments) is attached to
dystrophin at multiple sites: at the N-terminus, in the central rod domain, and at the C-terminus of the protein.

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Duchenne Muscular Dystrophy

dystrophin lacks the cysteine-rich region and the Cterminal domains. Recent transgenic animal studies show
that the cysteine-rich domain of dystrophin is critical for
interacting with DGC in vivo and that disruption of this
interaction renders dystrophin completely nonfunctional.
However, deleting the alternatively spliced, extreme Cterminus of dystrophin has no apparent eect on the
function of dystrophin. Furthermore, overexpression of
Dp71 (the truncated C-terminal region of dystrophin) in
transgenic mdx mice, an animal model of DMD, restores
the level of the DGC; however, it fails to prevent muscle
bres from degenerating.

DystrophinGlycoprotein Complex
Biochemical, molecular biological and immunological
techniques show that dystrophin binds to a large oligomeric complex of proteins and glycoproteins known as the
DGC. The proposed organization of the DGC is shown in
Figure 1. The DGC is formed from dystroglycan, sarcoglycan and syntrophin complexes.
Dystroglycan consists of a 156-kDa extracellular matrix
(ECM) laminin-binding subunit (a-dystroglycan) and a
43-kDa transmembrane subunit (b-dystroglycan). Both aand b-dystroglycans are derived from a common precursor
protein. The two proteins form a continuous link between
laminin-2 and dystrophin. a-Dystroglycan binds to laminin and b-dystroglycan; b-dystroglycan is connected to the
cysteine-rich region of dystrophin (Figure 1). It appears that
the DGC may have more than one function: a role in
communication between the ECM and cell interior, and/or
a role in cytoskeletal organization. At the neuromuscular
junction a-dystroglycan also associates with agrin, a
component of the synaptic basal lamina, and induces the
clustering of acetylcholine receptors.
Sarcoglycans are muscle-specic integral membrane
components of the DGC (Figure 1). The four sarcoglycans
identied to date are: a 50-kDa a-sarcoglycan, a 43-kDa bsarcoglycan, a 35-kDa g-sarcoglycan and a newly identied 35-kDa d-sarcoglycan (Figure 1). Sarcoglycans form a
tight complex; they have a large extracellular domain and a
relatively short cytoplasmic region. The muscular dystrophies arising from mutations in a-, b- and g-sarcoglycans
are now classied as limb girdle muscular dystrophy
(LGMD) types 2D, E and C, respectively (Table 1). The
gene responsible for LGMD2A encodes for calpain-3, a
calcium-dependent protease.
LGMD is characterized by loss of the entire sarcoglycan
complex from the sarcolemma membrane. One sarcoglycan may destabilize the complex and/or increase susceptibility to proteolysis during biosynthesis and assembly. A
novel component of the sarcoglycan, a 35-kDa d-sarcoglycan, was recently identied. Mutations in the d-sarcoglycan gene are responsible for LGMD2F. To date there is no

evidence for direct sarcoglycandystrophin interactions,

although sarcoglycans may interact with a-dystroglycan
and contribute to DGC function (Figure 1). This complex is
likely to be involved in a signalling pathway, further
supporting the hypothesis that DGC is involved in cellular
signalling.
In the neuromuscular junction sarcolemma agrin, a
component of the synaptic basal lamina, binds to adystroglycan and induces the clustering of acetylcholine
receptors (AChRs). Recent work from several laboratories
suggests, however, that a-dystroglycan may play only a
secondary role in AChR aggregation. Agrin-induced
AChR aggregation requires tyrosine phosphorylation
and may involve the receptor tyrosine kinase, suggesting
that several membrane-associated proteins may be involved in agrin-dependent clustering of AChRs. Laminininduced and tyrosine phosphorylation-independent aggregation of dystroglycan leads to the formation of dystroglycan and dystrophin-containing clusters. This is the rst
evidence for a potential role of laminin in the mobilization
of dystroglycan to assemble into the functional DGC.
In general, mutational analysis of patients with DMD
and BMD suggests that both the N- and C-terminal
domains of dystrophin are necessary for normal function
of the protein in skeletal muscle, and a mutation or deletion
that results in the loss of any component of these terminal
domains results in DMD. Conversely, the a-helical domain
of the protein can withstand large in-frame deletions and
still result in an apparently semifunctional dystrophin and
mild BMD. In summary, defects in dystrophin leading to
loss of attachment to the ECM or the internal cytoskeleton
result in DMD. Defects in dystrophin that result in inframe loss of a large proportion of the a-helical segment
result in the milder BMD.
Neuromuscular disorders and/or cardiomyopathies
consist of a wide spectrum of diseases of varying aetiology.
Many of them are not well understood with respect to the
primary defects underlying the molecular changes leading
to cellular abnormalities in muscle bre. Besides insight
into the molecular pathogenesis of DMD and BMD,
elucidation of the interaction of dystrophin with other
components of the muscle bre has led to the discovery of
primary defects leading to other related neuromuscular
disorders. Table 1 shows that defects in dystrophin and/or
other components of the DGC are responsible for several
phenotypes of muscular dystrophy including DMD,
BMD, severe childhood autosomal recessive muscular
dystrophy and LGMD.
In conclusion, abnormalities in components of the DGC
appear to be the underlying molecular cause that triggers
muscle degeneration in many muscular dystrophies.

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Duchenne Muscular Dystrophy

Table 1 Selected human muscular dystrophies

Type of muscular dystrophy

Protein product

Locus

X-linked recessive inheritance

Duchenne muscular dystrophy
Becker muscular dystrophy
EmergyDreifuss muscular dystrophy

Dystrophin
Dystrophin
Emerin

Xp21
Xp21
Xq28

Autosomal recessive inheritance

LGMD2A
LGMD2B

Calpain-3
Not known

LGMD2C
LGMD2D
LGMD2E
LGMD2F

g-Sarcoglycan
a-Sarcoglycan
b-Sarcoglycan
d-Sarcoglycan

15q15
2p1214
13q12
17q12
4q12
5q3334

Autosomal dominant inheritance

LGMD1A

Not known

5q

LGMD, limb girdle muscular dystrophy.

Function of Dystrophin

Therapies

Based on a vast body of experimental and clinical ndings

in DMD, at least three main hypotheses exist to explain
how muscle bre degeneration is initiated. While the
mechanical hypothesis suggests a direct link between the
lack of dystrophin and the mechanical fragility of the
muscle bre, the calcium inux hypothesis is based on the
assumption that an abnormal accumulation of intracellular calcium is caused by dystrophin deciency. A third
option is the membrane-associated signalling hypothesis,
which proposes that any changes in the intracellular
environment due to perturbation of membrane-associated
signalling mechanisms might result in muscle bre
destruction. The analysis of the DGC complex in DMD
muscle would support mostly the mechanical hypothesis:
the disruption of the linkage between the ECM and the
subsarcolemmal membrane cytoskeleton might render
muscle more susceptible to necrosis. However, the
characteristic multiple stages of the disorder argue against
a role of mechanical instability in the damage seen in
dystrophin-decient muscle. The balance of data available
indicates that dystrophin-decient muscles do not have
any inherent defect in plasma membrane permeability.
However, once muscle degeneration has been initiated,
both inux of calcium and eux of creatine kinase occur.
Clearly, further information on the pathological processes
in skeletal muscle is required in order to evaluate fully the
relevance of these hypotheses to the pathophysiology of
DMD and BMD.

Several research groups have been developing myoblast

transfer techniques in an attempt to replace the defective
gene in dystrophic skeletal muscle. Myoblasts are muscle
precursor cells capable of migrating to the site of muscle
damage, fusing with other myoblasts and thus regenerating
the muscle. The aim of myoblast transfer is to incorporate
donor myoblasts (containing dystrophin) into dystrophindecient tissue. These would fuse with the existing muscle
bres and thus incorporate dystrophin. Initial experiments
in mdx mice were promising, but ecient transfer of
myoblasts into human muscles has not yet been successful.
One of the major problems is that cardiac muscle does not
regenerate and thus myoblast transfer is not feasible in this
tissue.
Gene therapies are being developed for a number of
disorders. Many researchers believe that DMD is an
incurable disease and that gene therapy may not provide an
immediate solution. However, the latest ndings appear
very promising and suggest that an upregulation of
dystrophin-like protein (utrophin) has benecial eects,
at least in animal models. Mdx mice overexpressing
utrophin exhibit raised levels of utrophin, reduced
dystrophin pathology and a restoration of all the
components of the DGC. Serum creatine kinase levels
also return to normal in these animals, indicating that the
restored DGC is functional. These exciting results provide
a new and promising avenue to DMD therapy. The key
question is what controls utrophin gene expression. Recent
investigation of molecular mechanisms of regulation of
expression of the utrophin gene identied nerve-derived

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Duchenne Muscular Dystrophy

factors that may be involved in transactivation of the gene.

This knowledge may be essential for the upregulation of
utrophin as a potential therapeutic strategy for DMD. In
the future, new drugs may be developed to target the
utrophin gene to increase the specic expression of this
protein to compensate for the loss of dystrophin.

Conclusions
Signicant progress has been made in understanding the
molecular organization of the DGC through molecular
cloning of new sarcoglycan, syntrophins and DRP2.
Research on the structure and function of dystrophin
and its associated glycoproteins should further advance
our understanding of the molecular pathogenesis of
muscular dystrophies. This will be critical for development
of new approaches to DMD therapy.

Further Reading

Boyce FM, Beggs AH and Kunkel LM (1991) Muscular dystrophy

research: what have we learned and where do we go from here? In:
McHugh PR and McKusick VA (eds) Genes, Brain, and Behavior, pp.
121127. New York: Raven Press.
Campbell KP (1995) Three muscular dystrophies: loss of cytoskeletonextracellular matrix linkage. Cell 80: 675679.
Fabrizio E, Pons F, Robert A et al. (1994) The dystrophin superfamily:
variability and complexity. Journal of Muscle Research and Cell
Motility 15: 595606.
Karpati G (1997) Utrophin muscles in on the action. Nature Medicine 3:
2223.
Ohlendieck K (1996) Towards an understanding of the dystrophin
glycoprotein complex: linkage between the extracellular matrix and
the membrane cytoskeleton in nuscle bres. European Journal of Cell
Biology 69: 110.
Tinsley JM, Blake DJ, Zuellig RA and Davies KE (1994) Increasing
complexity of the dystrophin-associated protein complex. Proceedings
of the National Academy of Sciences of the USA 91: 83078313.
Winder SJ (1997) The membranecytoskeleton interface: the role of
dystrophin and utrophin. Journal of Muscle Research and Cell Motility
18: 617629.
Worton R (1995) Muscular dystrophies: diseases of the dystrophin
glycoprotein complex. Science 270: 755756.

Ahn AH and Kunkel LM (1993) The structural and functional diversity

of dystrophin. Nature Genetics 3: 283291.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net