Special Article: The Bernard Sachs Lecture
The Duchenne Dystrophy Story:
From Phenotype to Gene and
Potential Treatment
Victor Dubowitz, MD, PhD, FRCP
and in particular the last
The past decade,
year, has seen an avalanche of interest and
advance in Duchenne dystrophy, and I thought this
an opportune time to look at the disease from its
historical roots right through to the isolation and
sequencing of the gene and the recognition of the
protein it transcribes, and to speculate on patho-
genesis and potential treatment.
Historical View
Eponymous titles are usually the prerogative of the
second person to describe a disease, and Duchenne is
no exception. He described his first case of muscular
dystrophy in 18611 under the title &dquo;paraplegie hyper-
trophique de 1’enfance de cause c6r6brale&dquo; in view of
the associated intellectual impairment. Some 10 years
earlier a London physician, Meryon, had documented
in lucid detail eight affected boys in three families.22
Credit for the first report, however, probably belongs
to the Italians Conte and Goija, who in 1836 recorded
two affected brothers at the Ospedali degli Incurabili
in Naples.33
In a series of further case reports in 1868,44
Duchenne drew attention to the striking prominence
of the muscles and coined the term &dquo;pseudohyper-
trophic,&dquo; which was taken up by Gowers in 1879 as a
descriptive title in his series of lectures on pseudo-
hypertrophic muscular paralySiS5 and remained firmly
entrenched as a diagnostic label until comparatively
recently. Duchenne also drew attention to the marked
proliferation of connective tissue in the muscle, which
he was able to study in muscle biopsies obtained with
Received May 24, 1989. Accepted for publication June 13,
1989. Based on 1988 Bernard Sachs lecture to the American Child
Neurology Society, Halifax, Nova Scotia, Sept 15, 1988.
From the Department of Paediatrics & Neonatal Medicine
and the Jerry Lewis Muscle Research Centre, Royal Postgraduate
Medical School, Hammersmith Hospital, London, England.
Address correspondence to Dr Dubowitz, Hammersmith
Hospital, Du Cane Road, London W12 ONN, England.
240
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the
to
needle-harpoon he devised. As an alternative title
&dquo;paralysie musculaire pseudohypertrophique&dquo; he
suggested &dquo;paralysie myosclerosique.&dquo; The term
muscular dystrophy was first coined by Erb in 1891.6
The more benign Becker form of X-linked mus-
cular dystrophy, was described by Becker and Kiener
in 1955~ in several members of Kiener’s own family,
and further two families were documented in 1962
a
by Becker,8 who thought the condition might be
allelic to Duchenne dystrophy. Clinically, it looks like
a photocopy of Duchenne dystrophy, apart from its
later onset and slower course.
The Phenotype
The vast majority of boys with Duchenne dystrophy
have lost the ability to walk by the age of 12 years. If
one defines Becker dystrophy as being ambulant
beyond 16 years of age, this provides a clear-cut
separation of the two phenotypes. Inevitably, there
will also be some intermediate cases who stop walking
between 13 and 16 years of age. There is marked
clinical variability within Becker dystrophy, with the
more severe cases losing ambulation in their late
teens and the more benign ones continuing ambu-
lation beyond 20 years of age and even into late adult
life. There is also variability within the Duchenne
group, some having a more rapid loss of power, with
inability to walk as early as 6 or 7 years of age, in
contrast to a median of about 10 years of age and the
more slowly progressive case who walks until 12 years
of age. This variability does not appear to correlate
with the presence or degree of associated intellectual
impairment.
Intellectual impairment is a consistent feature of
Duchenne dystrophy, although only about 20% to
30% actually fall in the subnormal range (IQ < 70).
Detailed psychometric assessment in several large
series showed a mean IQ in the region of 85, with
a range from about 40 to 130. The distribution is
Gaussian and not bimodal, so that the most likely
 to lower values,
interpretation is a general shift in IQ
rather than selective involvement in only some cases,
with a separate genetic basis.9no The intellectual
impairment is nonprogressive, verbal ability is more
severely affected than performance, and there is
usually concordance in mental ability in affected boys
within a family.
There is also consistent involvement of cardiac
muscle in Duchenne dystrophy. In the early stages,
~
this is reflected by electrocardiographic changes, but
’
in the late phases, there may be overt signs of cardiac
decompensation and failure. At autopsy, the cardiac
muscle shows dystrophic change, often accompanied
by extensive fibrosis.
The involvement of smooth muscle has been
more tenuous and has rested on the occasional case
reports of acute gastric dilation or bladder paralysis,
usually as a terminal event. There have also been
autopsy reports of changes in the smooth muscle of
the bowel wall, and a recent clinical study suggested
abnormal emptying of the stomach. 11
Theories on Pathogenesis
Since the first authors, there has been speculation on
the pathogenesis of muscular dystrophy. In recent
years three hypotheses have been in vogue: (1) the
vascular hypothesis, based on the assumption that
the necrosis of the muscle fibers may result from
ischemia; (2) the neurogenic hypothesis, suggesting
an abnormality in the trophic influence of nerve on
muscle; and (3) the membrane hypothesis, which
postulates a primary abnormality in the cell membrane.
The membrane theory has been widely sup-
ported since the observation of grossly elevated levels
of enzymes in the serum in Duchenne dystrophy.
Further support came from the observations with
electron microscopy of breaks in the membrane and
focal lesions, the so-called delta lesions,12 and from
changes in intramembranous particles observed in
freeze-fracture studies of the muscle membrane. A
membrane abnormality could also be linked with the
possible role of increased intracellular calcium con-
centrations in Duchenne dystrophy in causing mus-
cle necrosis by enhancement of calcium-activated
proteases and mitochondrial calcium overload, re-
sulting in a reduction in oxidative phosphorylation
and eventual cell dearth. 13 A lectin-binding membrane
protein has been shown in our laboratory to be absent
in Duchenne dystrophy, but present in normal
muscle and other neuromuscular disorders. 14 This
glycoprotein is present in the muscle of fetuses at
high risk of Duchenne dystrophy but disappears
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postnatally in the course of the disease, suggesting
that it is a secondary event relating to the dystrophic
process. 15
A role for various autoimmune pro-
possible
cesses or the progression of the
in the initiation
dystrophic process have recently been highlighted
by the demonstration of T cells in the infiltrates of
round cells in dystrophic muscle.&dquo; An abnormal
component within the muscle fiber could produce a
change in surface cell antigens, which cytotoxic
lymphocytes then recognize as nonself, causing
them to attack the muscle membrane. A similar
mechanism could be associated with the observations
that HLA class I antigens, which are not expressed in
normal skeletal muscle, are expressed in Duchenne
and Becker dystrophies and also in polymyositis and
may render the muscle fibers susceptible to T-cell
attack
All these activities moved to the sidelines as the
new techniques of &dquo;reversed genetics&dquo; succeeded in
locating and identifying the defective Duchenne
gene, without prior knowledge of the gene product.
The Duchenne Gene
Localization of the Gene
The exact location of the gene for Duchenne dys-
trophy came from three different sources. In the first
place, several reports appeared of females with an
apparently classical Duchenne-type dystrophy, all of
whom had a reciprocal translocation between the X
chromosome and an autosome. Different autosomes
were involved in the translocations, but in each
instance the break-point on the X chromosome was
in the region of Xp2l. In X:autosome translocations,
the normal X is preferentially inactivated so that
genes on the derived X are expressed. One could
thus assume that the mutant gene for Duchenne
dystrophy is located at Xp21. Some 20 such cases
have now been reported and although they were all
described as Duchenne-type dystrophy, some had a
milder phenotype. 18,19
The second source was the discovery of DNA
markers linked to the Duchenne gene. Through the
application of the new techniques of molecular gen-
etics, it became possible to isolate DNA sequences
from cloned fragments derived from the human X
chromosome and to determine their exact location. 20,21
Duchenne dystrophy was the first disorder to be
localized in this way with restriction fragment length
polymorphisms (RFLPs). In a parallel study of Becker
muscular dystrophy, similar linkage was found to
241
these RFLPs,22 supporting the view that Duchenne
and Becker dystrophies are allelic.
The third source of localization was from the
documentation of a remarkable boy with Duchenne
dystrophy who also had chronic granulomatous
disease, retinitis pigmentosa, and McLeod syndrome
(lack of Kell antigen in red cells, acanthocytosis, and
raised creatine kinase levels). High-resolution chro-
mosome banding showed a small interstitial deletion
in the Xp21 band, which presumptively included the
Duchenne gene.23
Isolation of the Gene
Two different strategies were used to try and isolate
the Duchenne gene. Worton and his coworkers
isolated the junctional region from the Duchenne
female with an X:21 translocation documented by
Verellen-Dumoulin et al. 24 They cloned the region
spanning the translocation break-point, which pre-
sumably contained at least part of the Duchenne
locus.25 A sequence derived from this clone (XJ1.1)
detected an RFLP closely linked to Duchenne dys-
trophy and also failed to hybridize with DNA from
some patients with Duchenne dystrophy, indicating
the presence of a deletion of the region complementary
to the probe.26
Kunkel and his colleagues2’ devised an ingenious
way of preparing a library of cloned sequences (the
pERT probes) corresponding to the DNA deleted in
the patient with the visible deletion of Francke et a1.23
These probes detected several RFLPs closely linked
with Duchenne dystrophy and also deletions in some
boys,28 which varied in length in different families.
In order to study a large enough cohort of patients
to obtain meaningful deletion data, Kunkel made the
pERT probes available to laboratories around the world
and was able to pool the data on some 1,201 cases of
Duchenne and 145 cases of Becker dystrophy and to
produce a paper with 74 coauthors.29 Initially it was
thought that only Duchenne cases showed deletions
with the pERT probes, but following the demon-
stration in one of our Becker patients3° of a large
deletion involving all three of the probes tested, other
reports of Becker cases with deletions followed. Ap-
plication of the pERT and XJ1.1 genomic probes
together with the earlier flanking probes have been
extremely valuable in more accurate carrier detection
in addition to serum creatine kinase, and in antenatal
diagnosis of the at-risk fetus based on chorionic villus
sampling early in pregnancy. The reliability of ante-
natal diagnosis is increased if the affected male in the
family has a deletion, since this can be recognized
unequivocally in a male fetus.
242
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Characterization of the Gene
Both the XJ and pERT probes have shown recom-
bination with Duchenne muscular dystrophy, in-
dicating that the gene is very large or involves a
region prone to frequent rearrangements. By chro-
mosome walking on either side of pERT 87, Kunkel’s
group was able to identify two small regions that
were highly conserved in several _animal species.31
These were used to search for transcripts in RNA
isolated from fetal tissues. From a transcript in fetal
muscle, a corresponding 14-kb CDNA was charac-
terized. The Duchenne gene was subsequently shown
to encompass at least 2,000 kilobases (2 million base
pairs) with a minimum of 60 exons spanning the
gene.32 This would be equivalent to about 0.05% of
the total human genome. The fact that Duchenne
DNA only encompasses 14 kb implies that less than
1 % of the total genomic DNA is eventually transcribed
into protein. It is not surprising that the complex
splicing procedure needed to remove the noncoding
introns should provide ample opportunity for &dquo;mis-
takes&dquo; to be made in the processing of the gene and .
the production of the protein product.
The CDNA clones have detected exon deletions
in the Duchenne locus with a frequency around
70%, compared with about 10% of deletions with
the genomic probes. This has considerably increased
the diagnostic potential in relation to carrier detection
and antenatal diagnosis, as well as the assessment of
isolated cases of Duchenne or Becker dystrophy and
the differentiation of them from autosomal recessive
limb-girdle dystrophy and spinal muscular atrophy.
Deletions of similar size may be associated with either
a Duchenne or a Becker type dystrophy. In Duchenne
muscular dystrophy, the deletions and the exons
involved are extremely heterogeneous, whereas in
Becker dystrophy they are more consistent and the
same deletions account for about 90% .33 Patients
who do not have a detectable abnormality with the
cDNA probes presumably have point mutations or
deletions too small to detect on Southern blots.
The Duchenne Protein
Given the large size of the Duchenne gene and the
anticipation of a protein in the region of 400 kd in size,
Wood et a134 speculated that the protein might be
nebulin, a cytoskeletal protein of appropriate size,
and indeed they were able to demonstrate its absence
in Duchenne biopsies they brought out of frozen
storage. However, the development of antibodies to
nebulin showed that it was still present in Duchenne
muscle35,36 and the gene for nebulin was subsequently
localized to chromosome 2. In addition, Hoffman et
al37 excluded nebulin as the Duchenne gene product
on the basis of the dystrophy CDNA sequence.
Hoffman et a138 identified the protein product of
the Duchenne muscular dystrophy locus by using
polyclonal antibodies directed against fusion proteins
coding for two distinct regions of Duchenne cDNA.
The protein was approximately 400 kd and represented
only about 0.002% of total striated muscle protein,
based on mRNA levels. It was also present in cardiac
muscle and, in much lower concentration, in smooth
muscle (stomach) and in mouse brain. The protein was
absent in the muscle from two boys with Duchenne
dystrophy. They named the protein dystrophin
because of its identification via the isolation of the
Duchenne muscular dystrophy locus.
Animal Models of Muscular Dystrophy
The mdx Mouse
An X-linked murine muscular dystrophy was dis-
covered purely by chance during the screening of
normal mouse serum enzymes in connection with a
mutagenesis screen.39 After a brief episode of severe
muscle degeneration at 2 to 3 weeks of age, the
muscles recover .40 The mice then remain essentially
normal with no overt weakness and a normal life
span.41 Bridges42 noted that, although the muscle
showed the degeneration and regeneration of dys-
trophic muscle, it did not manifest the connective
tissue proliferation characteristic of Duchenne dys-
trophy. The amino acid sequence of human and
mouse dystrophin has been shown to be greater than
90% homologous over the entire amino-terminal
third of the protein (approximately 130 kd). 37,43 In
addition, Hoffman, et al3g demonstrated the absence
of dystrophin in the mdx mouse, indicating that mdx
and Duchenne dystrophy probably represent the
same genetic disorder. Recent studies have con-
firmed that mdx is a mutation mapping in the mouse
dystrophin gene.44 The mdx mouse presents a fas-
cinating animal model for the Duchenne gene in view
of its genetic identity yet absence of the dystrophic
phenotype.
Canine X-Linked Muscular Dystrophy
A myopathy with X-linked inheritance has recently
been recognized in a strain of retriever dogs that
bears a striking phenotypic resemblance to Duchenne
dystrophy.45 A colony was established by breeding
an affected male with healthy females to produce
obligate carrier females. Clinical signs begin at about
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8 weeks of age, characterized by progressive weak-
ness and stiffness associated with limb contractures.
Although clinical signs were consistent within litters,
their severity often varied between affected individuals
from different litters. The histological picture is re-
markably similar to that of Duchenne dystrophy, and
perimysial and endomysial fibrosis may already be
present by 4 weeks of age. (During my 1988 summer
vacation at India Lake, PA, I took a day off to visit
Ithaca and review the clinical and histological fea-
tures of these dogs through the kind courtesy of Drs
Beth Valentine and Barry Cooper.)
In a study of the molecular genetic relationship of
canine and Duchenne dystrophies, Cooper et al46
were able to demonstrate that the 14-kb transcript
of the Duchenne gene and its protein product, dys-
trophin, were absent in the dystrophic dog and were
present at intermediate levels in the heterozygous
female. In the nondystrophic dog, the antibodies to
dystrophin detected a protein with a relative mass of
400 kd indistinguishable from that present in human
and mouse muscle. The canine dystrophy thus
provides an animal model with phenotypic as well
as genotypic identity with Duchenne dystrophy
(Figures 1 and 2). Because of its striking clinicopath-
ological resemblance to Duchenne dystrophy, it could
prove particularly useful in studying the pathogenesis
of muscle degeneration and fibrosis and in assessing
therapeutic strategies.
Dystrophin
Localization of Dystrophin
In their initial studies after the discovery of dys-
trophin, Hoffman et a148 found it to be concentrated
in a microsomal fraction on sucrose gradients, and
localized it to the triadic junction. They thought
it was probably involved with Ca2+ homeostasis.
However, subsequent immunocytochemical studies
showed its localization in the sarcolemmal membrane
of normal muscle and its absence in the muscle of
Duchenne dystrophy and the mdx mouse. 49-52
The distribution of dystrophin in different tissues
is still not fully resolved. Dystrophin messenger RNA
is difficult to study in both healthy and pathological
tissues because of its large size (14 kilobases) and low
abundance (0.01 % to 0.001 % of total muscle mRNA).
Using a highly sensitive amplification technique,
Chelly et al53 were able to demonstrate the presence
of a dystrophin-specific transcript (mRNA) in 13
different human tissues, including brain cortex, at a
concentration about 100 times less than skeletal
243
 FIGURE 1 _

A. A 4-year-old boy with Duchenne muscular dystrophy. He has difficulty running and
going up steps and gets up from the floor with a Gowers’ maneuver. B. A 14-year-old
boy with Becker muscular dystrophy. He is currently still ambulant at 20 years of age and
the progression of his weakness has been very slow. C. A 6-week-old mdx mouse with
no detectable clinical weakness. D. A 6-month-old dog with X-linked canine muscular

dystrophy, showing marked wasting of limb muscles and considerable disability. E. and
F. Canine Gowers’ sign, showing the difficulty a dystrophic dog has in getting up from a
supine position. (A and B reprinted from Dubowitz4~ with permission of Wolfe Medical
Publications.)

muscle. Nudel et a1,54 using a ribonuclease protection
assay, were also able to demonstrate the presence of
a Duchenne dystrophy gene transcript in rat and
mouse myogenic cell cultures, in rat and mouse
striated muscle, in mouse smooth muscle, and in rat,
mouse, and rabbit brain but not in other nonmuscle
tissues. Recently Nudel et a155 have demonstrated
subtle differences in the transcript of the Duchenne
gene and the amino-terminal of the encoded protein
in brain and muscle. The 5’ ends of these mRNA
species are derived from different exons, suggesting
that the two mRNA types are transcribed by different
promoters.
&dquo;
Structure of Dystrophin
Koenig et a156 have recently determined the complete
sequence of human Duchenne muscular dystrophy
cDNA and the corresponding amino acid sequence,
from which they have been able to define different
domains of the protein and suggest a preliminary
structure. The amino acid sequence of dystrophin
shares many features with the cytoskeletal proteins
spectrin and a-actinin.
Function of Dystrophin
With the tentative resolution of the location of dys-
trophin and its structure, it has become possible to
start speculating on its function in healthy muscle
and on the pathogenesis of the dystrophic process in
its absence. The general consensus at present points
to structural role in the cell membrane and possibly
a
some stabilizing effect on the integrity of the mem-
brane. In its absence, physical stresses on the myo-
fiber might lead to necrosis.
Specificity of Dystrophin
In their initial study of dystrophin in relation to
Duchenne and Becker muscular dystrophies and
other neuromuscular disorders, Hoffman et a15~
concluded that dystrophin was absent in the severe
Duchenne dystrophy, was present but of abnormal
molecular size in mild Becker dystrophy, and was
present in normal amounts and size in other forms of
muscular dystrophy. Some of their patients did not
quite fit into this neat categorization. One Becker case
had absence of dystrophin which they ascribed to the
presence of a deletion corresponding to the same
DNA region as the fusion proteins used in preparing
the antibodies. In a comparable study of our muscle
biopsies with the Hoffman antibodies, we also found
consistent absence of the protein in all five Duchenne
muscular dystrophy cases and variable levels in four
Becker muscular dystrophy patients, which seemed
to relate to the abundance rather than molecular
size.58 We also found dystrophin to be completely
absent in four of six fetuses at high risk for Duchenne
muscular dystrophy, and present in trace amounts in
the remaining two.
Using the same two Hoffman antibodies, we re-

244

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 .

FIGURE 2
A. Quadriceps muscle biopsy from case shown in Figure 1A, Duchenne dystrophy,
showing variation in fiber size, marked separation of fibers by endomysial connective
tissue profileration, and focal necrosis and phagocytosis of fibers. (H&E; original
magnification, X140) B. Quadriceps muscle biopsy from case shown in Figure 1B, Becker
muscular dystrophy, showing variation in fiber size with increase in size of many fibers,
internal nuclei, splitting of fibers, and focal necrosis of fibers. There is mild endomysial
connective tissue proliferation. (H&E; original magnification, X63) C. Extensor digitorum
longus muscle from 6-week-old mdx mouse, showing good preservation of muscle
bundles, mild variation in fiber size, and clusters of small fibers with central nuclei,
presumably regenerating. (H&E; original magnification, X140) D. Triceps muscle from
4-month-old dystrophic dog, showing marked variation in fiber size and endomysial
connective tissue proliferation, dark staining hypercontracted fibers, and large clusters of
necrotic/regenerating fibers. (Trichrome; original magnification, X63)

cently found complete absence of dystrophin in a
classic case of mild Becker muscular dystrophy; the
patient is now 20 years old and still freely ambulant. 59
In addition, he had no deletion present with the
cDNA probes covering the whole length of the gene.
Deletions of comparable size and location can
occur in Duchenne and Becker dystrophy. Monaco
et al6° postulated that in Duchenne dystrophy the
deletion, irrespective of size, may lead to a frame shift
of triplet codons for amino acids resulting in severely
truncated nonfunctional protein, whereas in Becker
dystrophy the nucleotides remain in frame, and a
functional protein can still be produced. However,
Malhorta et a161 have recently demonstrated a con-
sistent deletion of exons 3 through 7 in six Becker,
five intermediate, and two Duchenne muscular dys-
trophy patients that did disrupt the translational
frame. They have postulated mechanisms that could
compensate for the effects of the frame shift in the
milder cases. The status of the muscle dystrophin in
these individuals has not yet been studied.
In a review of 218 of our patients with Duchenne,

245

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Becker, or intermediate
phenotypes, 124 had dele-
tions with the cDNA probes. Seventy-four separate
deletions were identified, with 55 being unique to
one patient and the remaining 19 occurring in at least
two unrelated patients.62
Deletions of exons 33 to
34 and 33 to 35 occurred with Becker muscular
only
dystrophy patients, and deletion of exons 3 through
7 occurred in four patients with intermediate pheno-
type and one with Becker muscular dystrophy. There
was also no correlation of associated mental retar-
dation with any selective deletions.
We have also recently had the opportunity to
investigate three sisters aged 4 years, 2’/2 years, and
13 months with a typical Duchenne muscular dys-
trophy phenotype and normal chromosomes. Muscle
dystrophin was normal in all three. They are pro-
bably cases of limb-girdle dystrophy. Their parents
are cousins, and the inheritance is presumably auto-
somal recessive.
It thus seems possible that the complete absence
of dystrophin, as assessed with currently available
polyclonal antibodies, is usually associated with
severe Duchenne muscular dystrophy, but can
exceptionally also be associated with mild Becker
muscular dystrophy. It is also possible that a clinical
(and pathological) phenotype indistinguishable from
severe Duchenne muscular dystrophy can occur
through an autosomal recessive gene and have
normal dystrophin levels.
Unresolved Questions
There are a number of questions that still await
clarification in relation to the absence of dystrophin
and the pathogenesis of Duchenne dystrophy. Why
is the early development normal? Is the absence of
dystrophin merely an early trigger in the patho-
genesis of the dystrophy, with subsequent secondary
mechanisms being more important? Why is there
variability in the clinical severity? Does this possibly
reflect a better ability of the affected muscle to re-
generate and compensate for the necrotic process?
Why is there a selective pattern in the muscle in-
volvement although all muscles lack dystrophin?
Does this reflect the importance of secondary factors,
such as the relative stresses on the different muscle
groups, which are also operative and give a similar
distribution of weakness in unrelated neuromuscular
disorders such as the spinal muscular atrophies?
Why is the disease progressive? What is the mechanism
of the marked connective tissue proliferation in
Duchenne dystrophy, and does it possibly have a
246
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key role in the pathogenesis and progression of the
disease?
How does one account for the absence of any
weakness or progression in the mdx mouse, which
also lacks dystrophin? How does one explain the mild
case of the Becker allele which also lacks dystrophin?
Could the mdx mouse possibly represent a mild allele
comparable to this Becker case?
What other protein abnormalities may produce
an identical phenotype to Duchenne dystrophy in
females with an autosomal recessive disorder and
normal dystrophin? What is the role of other proteins
such as nebulin, which is reduced as a secondary
process in Duchenne and also various other dys-
trophies ? Does the lectin-binding membrane protein
found by Capaldi to be selectively lost in Duchenne
dystrophy have a key role in the pathogenesis?
To date, there has not been a report of a classic
Xp21 Duchenne dystrophy with the presence of dys-
trophin in normal or reasonable amounts. Possibly
this may never occur, but if such a case were to be
identified it would be another exception to the general
rule of Kunkel of correlating clinical severity with
absence or presence of dystrophin. It is also of interest
in this context that one of our isolated female cases
with a Duchenne-like phenotype, loss of ambulation
at 12 years of age, and normal chromosomes has
shown a low abundance of normal molecular weight
dystrophin and presumably represents a severely
manifesting Duchenne carrier.
Potential Treatment
Gene Therapy -
Although it may be exciting to speculate on the
possibility of gene therapy in muscular dystrophy, it
seems extremely unlikely that this could become a
reality in the foreseeable future. Techniques are
already available for gene transfer that have the
potential to correct genetic disorders by correcting
the abnormal gene itself. Defective genes can be
identified and isolated and normal copies can be
synthesised and transferred into cells. There are,
however, still major technical hurdles such as the
targeting of a new gene or its encoded protein to a
specific organ.
Protein Replacement
There also seem to be major barriers to the possibility
of replacing the deficient protein, given its large size,
its low abundance, and the difficulty of delivering a
relatively insoluble protein to the cell membranes.
Myoblast Implanation
One potentially viable approach would be the pro-
vision of normal precursor cells to affected muscle. A
number of experiments in recent years have shown
that this is already a feasible and potentially practical
approach. In a series of studies on the transplantation
of muscle between normal and dystrophic mice and
hamsters, primarily aimed at assessing the role of the
nervous system in the dystrophic process, we were
able to show, using isozymes of glucosephosphate
isomerase as a genetic marker, that heterotransplan-
tation of muscle mince between two different strains
of normal mice resulted in a regenerated muscle of
donor origin.63
Partridge and his colleagues 64,65 showed that
mononucleated cells prepared by enzymatic dis-
aggregation of neonatal mouse muscle could be
introduced into growing muscle fibers of young hosts
and that the resultant mosaic muscle fibers expressed
not only the glucosephosphate isomerase alleles
of the host and donor muscles but also a hybrid
isoenzyme dimer which could only result from fusion
of donor and host myoblasts in the same fiber. They
subsequently tried using this technique to correct the
defect in a strain of mice with inherited phosphorylase
kinase deficiency.66 Although myonuclei of donor
origin became incorporated in the regenerating muscle
fibers in eight of nine autografts, only three showed
phosphorylase kinase activity. Following injection of
normal muscle precursor cells into growing phos-
phorylase kinase-deficient skeletal muscle, mosaic
fibers with donor myonuclei were detected in only 11
of 192 muscles examined from 64 mice, but nine of
these contained phosphorylase kinase activity.
Law and his colleagues have conducted a series
of experiments over the past few years along similar
lines aimed directly at trying to improve the muscle
structure and function in mice with autosomal re-
cessive muscular dystrophy. 67 As the normal genome
is being incorporated into the dystrophic muscle
during myogenesis or regeneration, it is not necessary
to know which gene is responsible or what the nature
of the defect is. Cultured myoblasts from healthy
mouse embryos were injected into the right soleus of
20-day-old healthy or dystrophic mice, the other leg
serving as control. Hosts and donors were immuno-
compatible but had different genotype markers for
glucosephosphate isomerase. After six months, the
test dystrophic solei showed greater cross-sectional
area, total fiber number, wet weight, and twitch and
tetanus tensions than the control solei. They also had
a more normal histological appearance and better
fiber type distinction. The presence of the donor
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isozyme of glucosephosphate isomerase plus the
hybrid isozyme implied the survival and develop-
ment of donor myoblasts into normal myofibers and
fusion of normal myoblasts with dystrophic satellite
cells to produce genetically mosaic myofibers.
Partridge has recently extended his studies to the
mdx mouse. Injection of normal muscle precursor
cells into the tibialis anterior muscle of dystrophic
animals has produced muscle expressing the donor
or hybrid glucosephosphate isomerase isozyme. He
has also been able to demonstrate that these muscles
do express dystrophin, which is consistently absent
in the control muscles in the same animals.68
In the absence of clinical weakness, the mdx
mouse might not be a good model for assessment of
clinical therapeutic potential of this approach, and
the dystrophic dog with its phenotype more closely
resembling Duchenne dystrophy might provide a
better model. In this context it would also be of
interest to know what proportion of normal myo-
nuclei have to be present in the mosaic muscle of
X-linked female carriers in order to produce a normal
phenotype.
The main therapeutic hurdle with this approach
is how to deliver the normal precursor cells to a wide
range of muscles and not just focally by in situ in-
jection. To be feasible for treating the human disease,
this problem will have to be resolved. If that is
achieved, such a therapeutic approach could be
beneficial not only for Duchenne dystrophy but also
for any of the other forms of dystrophy, irrespective
of whether the inheritance is autosomal recessive or
X-linked recessive, and even in the face of complete
ignorance of the nature of the gene or the defect.
Another major hurdle is the problem of histocom-
patability and rejection of the implant. This can
readily be overcome in the mouse by producing
tolerance in the neonatal mice with injection of
fetal spleen cells. In the human and the dog, alterna-
tive approaches, possibly using immunosuppression,
would be necessary.
Other Stategies to Prevent Progression
For the present and foreseeable future, it seems likely
that we shall still have to look to other means of
intervention to try and halt the progression of the
disease, or at least attenuate its severity. Better in-
sight into the role of dystrophin in initiating the
dystrophic process and the role of other associated
proteins or other biochemical mechanisms in per-
petuating the process or causing its progression
could help in working out some rational approach
to treatment.
247
 We are also still uncertain as to the importance of
associated pathological processes, such as the pro-
liferation of perimysial and endomysial connective
tissue in the progression of the disease and the com-
petence of the regenerative potential of the muscle in
determining the severity.
Drug Therapy
Methods have been standardized for the evaluation
of potentially effective drugs, either in large multi-
centric studies69 or in smaller, more rigidly con-
trolled, unicentric studies .70 At present there is no
available drug with a proven effect on the course of
the disease. Under these circumstances, it seems
important to also look closely at physical means of
intervention, which may have an influence on the
course and progression of the disease.
Chronic Electrical Stimulation
In a study of the effects of chronic low-frequency
.
stimulation in the dystrophic mouse, Luthert and
colleagues found an improvement in function, and a
reduction in the loss of muscle fibers together with
increased levels of oxidative enzyme activity.
In a
comparable study of six young ambulant boys with
Duchenne dystrophy, chronic low-frequency stimu-
lation of the tibialis anterior (cycle: 1.5 seconds on,
1.5 seconds off, for one hour three times per day, at a
frequency of 5 to 10 Hz, for periods from 7 to 10
weeks) produced a significant increase in the maxi-
mum voluntary contraction of the stimulated legs
compared with the contralateral unstimulated control
legs .72A further study with chronic high-frequency
stimulation (30 Hz) in a separate group of six boys
with Duchenne muscular dystrophy produced a
reduction in the maximum voluntary contraction.
This suggests that chronic low-frequency stimu-
lation may be beneficial to the function of the muscles
in Duchenne dystrophy, and possible longer-term
stimulation of selected muscle groups could have a
therapeutic application.
Surgical Intervention
In the past few years,
Rideau et al have developed a
program of surgical interventions aimed at trying to
maximize the available muscle function in boys with
Duchenne muscular dystrophy in an effect to nor-
malize and prolong their activities in the earlier
phases of the disease?3 He initially released con-
tractures of muscles such as tensor fascia lata, ham-
strings, and tendo Achillis in relatively late ambulant
cases and gradually intervened at a younger age.
Currently he recommends the release of tight muscles/
tendons at the hips, knees, and ankles in the early
248
Downloaded from jcn.sagepub.com at The University of Auckland Library on June 4, 2015
stages of the disease, optimally between about 4 and
6 years of age, together with resection of the whole
fascia lata.
He claims that this has led to an improved ability
to rise from the floor and even loss of the Gowers sign
in some cases. The time these children take to get up
from the floor then remains stable for some three to
four years, without the usual progressive increase in
the time. They are also able to walk in a more normal
fashion on a narrow base without the customary
waddle and in some cases to run in a fairly normal
fashion, in contrast to the accentuation to the waddle
usually produced in boys with Duchenne muscular
dystrophy. There is, surprisingly, no recurrence of
the tightness of the tendo Achillis and no need for any
passive stretching. I was able to verify these obser-
vations in a personal visit to Professor Rideau’s unit
in November 1987. There was usually a remarkable
hypertrophy of the vastus lateralis muscle along the
line of incision for the fascia lata resection, almost
like a herniation or release from a compartment
syndrome.
Although long-term follow-up in these patients is
still limited, a point seems to be reached after a few
years of stability, when there is a rapidly progressive
increase in difficulty getting up from the floor and
subsequent loss of ambulation within a few months.
There number of mechanisms one can
are a
postulate explain the apparent improvement in
to
function following the surgical intervention. The
early contractures in the muscles may reflect the
progressive diffuse fibrosis in the muscle in the early
stages of the disease. Histologically, it is often already
quite striking by 3 years of age. These muscles them-
selves are compromised, but in addition the antagonist
muscles will be under continuous stretch, which may
have a damaging effect comparable to eccentric con-
traction under load in normal muscle. Release of
these contractures at an early stage, when the under-
lying power in the muscles is still good, may halt or
actually reverse the process and enable the muscles
to function optimally, given the limitations or con-
straints placed on the muscle by the primary under-
lying disease process.
I have set up a prospective, randomized, con-
trolled study to try and quantify the effects of this
surgical intervention and, by sequential ultrasound
imaging of selected muscles together with selective
needle biopsy, to identify a possible change or reversal
in the pathological pattern of connective tissue proli-
feration and other characteristic histological features.
Such clinical studies could well produce answers at
a biological level to some of the current questions
about the function of dystrophin and its relevance in
relation to the progressive dystrophic process, as well
as the role of other factors, which may possibly be
of more importance than dystrophin itself in per-
petuating the dystrophic process.
The past few years have been an exciting time in
relation to Duchenne dystrophy and have seen more
progress in our understanding of the underlying
abnormality than in the previous 150 years since
Conte and Goija’s first description. Hopefully, the
next few years may see an equally significant break-
through at the clinical therapeutic level.
Acknowledgments
Our muscle research program has been generously supported
by the Muscular Dystrophy Group of Great Britain, Muscular
Dystrophy Association of America, Medical Research Council,
Wellcome Trust and Action Research for the Crippled Child. I am
grateful to numerous colleagues for helpful discussions, to Barry
Cooper and Beth Valentine for access to the clinical and patho-
logical features of the dystrophic dog, to Terry Partridge for dys-
trophic mice, and to the Muscular Dystrophy Group of Great
Britain for their continued support of our muscle research program
in the Jerry Lewis Muscle Research Laboratories originally endowed
by the Muscular Dystrophy Association of America.
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