Abstracts from de Annual Meeting - American Academy of Neurology - april 2004 San Francisco
[P01.139] Bone Mineral Density in
Pediatric Neuromuscular Disorders. Are All Patients at Equal Risk of Developing
Osteopenia?
Ismail A. Khatri, Susan T. Iannaccone, Mouin G. Seikaly, Dallas, TX
OBJECTIVE: To evaluate bone mineral density (BMD) in different pediatric
neuromuscular disorders to identify the high-risk group(s) of patients. BACKGROUND:
Pathological fractures are common in pediatric neuromuscular disorders. The
relationship of BMD to fracture risk is well established in post-menopausal
women. However, this relationship is still controversial in children. Dual
energy X-ray absorptiometry (DEXA) has become a standard technique for the
measurement of BMD in adults. Relatively limited data is available on BMD in
pediatric neuromuscular diseases other than Duchenne muscular dystrophy. DESIGN/METHODS:
We reviewed the results of all DEXA scans done in our pediatric
neuromuscular clinic during 2002 and 2003. Bone mineral density was performed on
spine region L1-4 using dual X-ray absorptiometry (Hologic QDR 4500). Osteopenia
was classified as mild if the Z-scores were between 0 and -1.5; moderate if
Z-scores were between -1.5 and -2.5; and severe if Z scores were <-2.5. RESULTS:
84 DEXA scans were performed: 3 on patients with Becker muscular dystrophy (BEMD),
55 on patients with Duchenne muscular dystrophy (DMD), 3 on patients with limb
girdle muscular dystrophy (LGMD), 2 on patients with myotonic dystrophy (DM), 3
on patients with congenital myopathies (CM), 7 on patients with congenital
muscular dystrophies (CMD), and 11 on patients with spinal muscular atrophy
(SMA). The mean Z-scores +/- the standard error of the mean (SEM) in each group
were as follows: BEMD -1.53+/-0.40 (range -2.20 to -0.82); DMD -1.72+/-0.10
(range -3.91 to -0.11); LGMD -0.37+/-0.64* (range -1.22 to +0.91); DM
-0.25+/-0.05 (range -0.31 to -0.21); CM -1.77+/-1.13 (range -4.01 to -0.31); CMD
-1.67+/-0.42 (range -4.02 to -0.62); SMA -2.25+/-0.31* (range -3.82 to 0). ANOVA
showed that spinal muscular atrophy and limb girdle muscular dystrophy were
significantly different from the other disorders. CONCLUSIONS: 1.
Patients with spinal muscular atrophy have the lowest Z-scores and therefore may
be at high risk for pathologic fractures.
2. Children with limb girdle muscular dystrophies have high Z-scores and perhaps
are at minimal risk of pathological fractures.
3. We recommend that bone mineral density should be evaluated in all patients
with spinal muscular atrophy. Treatment for low BMD in children remains
controversial. Supported by: Muscular Dystrophy Association of America
P02.159] Strength and Fatigue in mdx
Mice Treated with Weekly Oral Prednisolone for 52 Weeks
Anne M. Connolly, Elizabeth M. Streif, Richard M. Keeling, St.
Louis, MO
OBJECTIVE: 1. To establish an effective in vivo measurement of strength
and fatigue in mdx mice. 2.To demonstrate long term effectiveness of
weekly oral prednisone in this mouse model. BACKGROUND: We have
previously shown weekly oral prednisolone prolongs survival and improves
strength in dydy mice(1). This regimen has also been shown to be
effective in one pilot study of boys with Duchenne muscular dystrophy (DMD)(2).
While there is debate about how well the mdx mouse mimics DMD, the
longest in vivo strength analysis was only 24 weeks(3). While corticosteroids
have been shown to benefit mdx mice over short periods of time, no long
term studies have been performed. Effective therapy of boys with DMD will
require lifelong treatment. Here we model treatment over one year in the C57Bl10
mdx mouse. DESIGN/METHODS: We studied forepaw grip strength 5
consecutive times at age 3, 4, 6, 8, 10, 12 and then every 4 weeks until 52
weeks. We calculated fatigue at each time point as previously described(3).
Eighteen mice were treated with weekly oral prednisolone (10mg/kg/week in two
doses on consecutive days); 18 were untreated. RESULTS: 1. While both
untreated mdx and controls peak strength by 8-10 weeks, the mdx is
significantly weaker at that time (17g.force/ g.body weight versus 20g.force/g.body
weight p<0.05). Thereafter, an average progressive decline in strength of 0.3
g.force/g.body weight occurs weekly (versus <0.1 g.force/g.body weight for
controls). 2. Mice treated with weekly oral prednisolone were stronger at each
time point and showed a slower average decline in strength (0.2g.force/g.body
weight). 3. Fatigue is present in all untreated mdx mice at all time
points (25-40% fatigue versus 0-5% for healthy controls p<0.005). 4.
Treatment with weekly oral prednisolone improved fatigue in female mdx at
ages 8-36 weeks (p<0.05) but does not become normal. Males show no
improvement. CONCLUSIONS: We demonstrate effective methods for measuring
long term strength and fatigue in the mdx mouse. We show for the first
time long term benefit of oral prednisolone in this model. Given that
corticosteroids remain the best treatment of boys with DMD and that multiple
pharmacologic agents may be necessary to improve outcome, it is critical to be
able to demonstrate in an animal model which drugs are acting synergistically or
antagonistically over long periods.
1. Connolly AM, Keeling RM, Streif E, Pestronk A, Mehta S. Complement 3
deficiency and oral prednisolone improve strength and prolong survival of
laminin α2-deficient mice. J Neuroimmunol 2002;127:80-87.
2. Connolly AM, Schierbecker J, Renna R, Florence J. High dose weekly oral
prednisone improves strength in boys with Duchenne muscular dystrophy.
Neuromuscul Disord 2002;12(10):917-925.
3. Connolly AM, Keeling RM, Mehta S, Pestronk A, Sanes JR. Three mouse models of
muscular dystrophy: the natural history of strength and fatigue in dystrophin-,
dystrophin/utrophin-, and laminin α2-deficient mice. Neuromusc Disord
2001;11:703-712. Supported by: Muscular Dystrophy Association
[P05.134] Testing Stabilizing Function
of Dystrophin in Human Duchenne Muscle Dystrophy Cells (DMD) vs. Control
Cells (CC) Due to Lowered Stress Tolerance: Promising In Vitro Testing
Tool for Possible Functional Vital Transfection Efficiency Measurements
Denis Bron, Sima Dadelahi, Ashley Hayes, Denise Brenklé, Friedel
Wenzel, Steck Andreas, Basel, Switzerland
OBJECTIVE: DMD is a X-linked myopathy and is caused by mutations in the
dystrophin encoding gene. As generally known, the absence of dystrophin causes
muscle fibre necrosis in DMD patients for example due to lowered stress
tolerance by membrane fragility. BACKGROUND: Current results in mouse
myotubes support suggested mechanical role of dystrophin in the context of the
membrane-cytoskeletal complex. To support the results in mouse myotubes, we
tested human DMD cell resistance due to osmotic stress. Further, we evaluated
these results for possible functional consequences of in vitro
transfection methods. DESIGN/METHODS: Human non transfected DMD- and
control- muscle cells were stressed by hypoosmotic Dulbecco buffer (100mosm).
Pictures were taken before buffer exposure and after 3 and 12 minutes.
Transcellular cell diameter measurements were taken and analysed statistically.
In addition, fluorescent enzymatic probes such as EthD-1 (intranuclear cell
death marker) and Calcein AM (cytoplasmatic vitality marker) were used as an
cell membrane and nuclear permeability marker. RESULTS: Statistically
significant differences in cell swelling comparing each cell line (DMD p=0.044,
CC p=0.43) were found after hypoosmotic exposure. The difference between each
cell was significant (p<0.001) higher in the DMD cell line. Further, the DMD
lysis rate differs statistically significant (p<0.0001) compared to CC. CONCLUSIONS:
Our results confirmed the mechanical role of dystrophin in human DMD cells.
Further, our in vitro study using the same cells demonstrates osmotic
resistance as promising tool testing cell transfection functionally. Combining
markers such as cell diameter measurements, lysis rate and fluorescent stains
increase diagnostic relevance. Further work is underway studying additional
parameters such as bleb manifestations and cytoplasmatic granulation.
[P01.118] Creatine Monohydrate
Supplementation Appears Safe for Children with Neuromuscular Disorders
Jonathan B. Strober, San Francisco, CA
OBJECTIVE: To evaluate the safety and efficacy of creatine
supplementation in a select cohort of children with neuromuscular disorders. BACKGROUND:
Creatine, muscles main store of energy, is transported in the blood by a
carrier protein and actively taken up by tissues with high energy demands with
95% of the total body creatine found in skeletal muscle Creatine supplementation
has been shown to raise its plasma concentration, sustainable with repeated
dosing, and can increase the total creatine content of muscles. Patients with
neuromuscular disorders have been found to have reduced concentrations of
phosphocreatine, a marker for total creatine. In two reports, renal dysfunction
has been exacerbated in patients receiving creatine supplementation. However, in
several studies of creatine supplementation in neuromuscular disorders no
significant side effects were reported. Some of these studies found mild, but
significant, benefit from supplementation, however, others found no benefit.
Therefore, continued investigation is warranted in children with neuromuscular
disorders. DESIGN/METHODS: Five children with neuromuscular disorders (2
with Duchenne muscular dystrophy (DMD), 2 with limb-girdle muscular dystrophy
and 1 with CIDP) were enrolled in this cohort study, 2 males (DMD) and 3 females
with an age range of 6 – 10 years. Patients took an initial loading dose of
creatine monohydrate 25 mg/kg/dose four times a day (100mg/kg/day) for a total
of 5 days, followed by 50 mg/kg/day in one daily dose for 30 days. Each child
underwent baseline testing, repeat testing at the end of the 30 days of
supplementation and one month off creatine. Strength was tested using myometry
and functional assessment was made using Jebsen hand-grip test and the Gross
Motor Function Measure. Muscle mass was determined by urine 3-methylhistidine
levels. To monitor for toxicity a complete blood count, serum electrolytes,
serum glucose and calcium, liver function tests, blood urea nitrogen and
creatinine, routine urinalysis and 24 hour urine creatine levels were obtained.
Toxicity was assessed using a modified common toxicity criteria from the Cancer
Therapy Evaluation Program. Any toxicity graded 3 or higher was considered
unacceptable. RESULTS: All five children completed the study without
significant side effects. The only child with any significant improvement was
the girl with CIDP, who showed a 200% increase in hip extension, sustained over
the 2-month testing period. Three of the patients with muscular dystrophy showed
a 25-50% decrease in hip flexion strength and one boy with DMD showed a 50%
decrease in hip abduction over the 2 months. All other measurements were not
significantly changed. CONCLUSIONS: While creatine monohydrate appears
safe in children with neuromuscular disorders, preliminary data suggest no
short-term benefits in muscle mass, gross motor function or strength. Supported
by: This study was funded in part in the UCSF Pediatric Clnical Research Center
with funds provided by the National Center for Research Resources M01RR01271 and
the UCSF
[P05.135] Missense Mutations in the Actin
Binding Domain (ABD) of Dystrophin Cause Muscular Dystrophy by Disrupting
Protein Stability
Fiona L. Norwood, Southampton, Hampshire, United Kingdom, Andrew
Sutherland-Smith, John Kendrick-Jones, Cambridge, United Kingdom
OBJECTIVE: To determine the effects of pathogenic mutations on dystrophin
protein stability. BACKGROUND: Dystrophin is a large cytoskeletal protein
whose absence or disruption is associated with Duchenne (DMD) or Becker muscular
dystrophies (BMD) respectively. The N-terminal domain of dystrophin has been
shown to bind to F-actin in vitro and in vivo and is termed the
actin binding domain (ABD). Several pathogenic mutations have been detected
within the human ABD of dystrophin. DESIGN/METHODS: Wildtype ABD
GST-fusion protein constructs were expressed, purified using affinity
chromatography, cleaved to remove their GST tags and their solubility and actin
binding function assessed. Further constructs in which the two cysteine residues
in the wildtype ABD were replaced by serine residues were made and the
functional assays repeated. Mutant ABD GST-fusion protein constructs were made
and the relative solubility of each expressed protein was assessed. RESULTS: Expression
and characterisation of the human dystrophin ABD led to the observation that
when the two cysteine residues normally present in this domain were replaced by
serines it greatly enhanced the subsequent purification and stability of the ABD
while preserving its ability to bind to F-actin. The introduction of pathogenic
mutations, associated with specific clinical phenotypes in muscular dystrophy
patients, into the ABD affected its ability to fold correctly and thus to be
purified and its properties tested. Furthermore, incorporation of these
pathogenic mutations into the cysteine exchanged wildtype ABD showed that these
single residue substitutions still caused the expressed domain to be unstable
and not to correctly fold. CONCLUSIONS: We predict that these pathogenic
residues must be in crucial positions in the actin binding domain so that when
mutated they destabilise its structure. Our structural analysis of this domain
has confirmed this conclusion. Supported by: Medical Research Council Laboratory
of Molecular Biology, Hills Road, Cambridge, UK.
[P02.157] Combined Deficiency of Calpain
and Dystrophin Mutually Reduce the Severity of Phenotypes?
Maria J. Molnar, Michael Sinnreich, Agnes Herczegfalvi, Eva Siska,
Budapest, Hungary, George Karpati, Montreal, QC, Canada
OBJECTIVE: To determine the effect of an abnormal dystrophin and calpain
expression profile on the skeletal muscle phenotype in patients with muscular
dystrophy. BACKGROUND: Dystrophin deficiency is an X chromosomal
inherited disease causing Duchenne/Becker muscular dystrophy. Calpainopathy is a
recessive disorder usually resulting in limb girdle muscular dystrophy. Both
gene defects usually result relatively severe phenotypes. DESIGN/METHODS: The
40 years old proband had mild quadriceps weakness and myalgia since age 32 years.
EMG revealed myopathic changes, serum CK activity was x 15 times elevated. His
sister is asymptomatic with serum CK 329U/l. The son of her sister was a clumsy
child and at age 6, mild leg weakness was observed. By age 10, his signs did not
progress. His serum CK activity is x 20 elevated. RESULTS: Histological
examination of the proband's muscle showed moderate dystrophic changes.
Immunocytochemical staining with Novocastra DYS1 and DYS3 antibodies revealed
only a few revertant fibers while with DYS2 antibodies, all fibers were
dystrophin positive. Western blot analysis of the muscle showed total absence of
calpain and a reduced size and amount of dystrophin shown with the DYS2 antibody,
confirming the diagnosis of Becker dystrophy and calpainopathy. Mutational
analysis of the dystrophin and calpain genes is in progress. CONCLUSIONS: This
is the first report of a patient with combined dystrophinopathy and calpain
deficiency. The unusually mild phenotype suggests that the combined deficiency
of calpain and dystrophin may mutually reduce the severity of phenotypes. This
observation might have an important impact on the molecular therapy of the
muscular dystrophies.
[EV.013] Factors Influencing the
Transduction Efficiency and Duration of Transgene Introduced into Muscles by
Plasmid-Mediated Electrotransfer (PMET)
Renald Gilbert, Nancy Larochelle, Yifan Lu, Maria J. Molnar,
Budapest, Hungary, An-Bang Liu, Hualien, Taiwan, Basil J. Petrof, Kristian
Orlopp, Hanns Lochmuller, Munich, Germany, Josephine Nalbantoglu, George Karpati,
Montreal, QC, Canada
OBJECTIVE: To determine factors that influence the level and duration of
transgene expression in muscle fibers in dystrophin-deficient (mdx) and of
immune incompetent (SCID) mice after PMET. BACKGROUND: Plasmid-mediated
gene transfer into muscle is a potentially safe and cost-effective procedure to
treat genetic deficiencies such as Duchenne muscular dystrophy (DMD). The
efficiency of plasmid-mediated gene transfer can dramatically be increased by
the application of an electric current (PMET) to the target muscle. One of the
unclear aspects of PMET into muscle fibers is the level and duration of
transgene expression in the presence versus absence of immune competency of the
host, as well as the role of isogenicity and size of the transgene. DESIGN/METHODS:
The tibialis anterior muscle (TA) of adult SCID mice was pretreated with
hyaluronidase followed by the electrotransfer of 30 μg of plasmid (pCBLacZ)
coding for β-galactosidase (β-gal). The TA of adult mdx mice was
treated in the same manner with pCBLacZ and with plasmids coding for the human
microdystrophin (9-kb, pHDysMic) or the murine full-length dystrophin (19-kb,
pMDysFl). The number of transduced fibers per muscle (β-gal+ or dys+) was
analyzed at 10, 90, 180 and 360 days post-treatment. In the case of SCID mice,
the amount of β-gal produced and the amount of plasmid DNA in the muscle
were also measured by luminometry and real time PCR, respectively. RESULTS: At
10 days post-injection, SCID muscle was transduced much more efficiently (1981
β-gal+ fibers) than mdx muscle ( 312 β-gal+ fibers) after PMET of
pCBLacZ. In mdx muscle, more muscle fibers were transduced using small plasmids
such pCBLacZ and pHDysMic (505 dys+ fibers) compared with pMDysFl (148 dys+
fibers). Although no significant reduction in the number of β-gal+ fibers
was observed in the SCID up to 360 days post-treatment, there was a reduction
(2.8X) of the amount of β-gal produced at 360 days. A significant reduction
(12X) in the amount of plasmid DNA in SCID muscle was demonstrated at 180 days
post-treatment. In mdx muscles injected with pMDysFl, the number of dys+ fibers
was also reduced at 180 ( 51) and 360 days (41) post-treatment. CONCLUSIONS: Transduction
efficiency of PMET varies greatly with the mouse strain and the transgene size.
Furthermore, even in the absence of a significant immune competency of the host
(SCID), there is a slow but significant decline of the plasmid DNA and transgene
protein level. This implies that repeated but relatively infrequent
administration of plasmid DNA may be necessary for continued high-level
production of therapeutic gene product in muscle.
[P02.156] Gene Expression Profiling in
Duchenne Muscular Dystrophy Patients Using Specifically Dedicated
Oligonucleotide Microarray
Armelle Magot, Martine Le Cunff, Nolwenn Le Meur, Jean Marie Mussini,
Jean J. Leger, Yann Pereon, Nantes, France
OBJECTIVE: To examine the pathogenic pathways and identify new or
modifying factors involved in Duchenne Muscular Dystrophy (DMD) using microarray
technology with muscle specific oligonucleotide chips. BACKGROUND: The
development of DNA microarrays for comprehensive RNA expression analysis raises
the exciting opportunity to examine biological pathways and to compare the
hypotheses deduced from the study of histological pathology with the findings of
molecular pathology. In particular, although the histopathological pathology of
dystrophic tissue is well documented, the underlying molecular pathways remain
poorly understood. DESIGN/METHODS: Skeletal muscle mRNA from four DMD
patients was compared with normal skeletal muscle mRNA. We developed a muscle
specific microarray comprised of 3588 oligonucleotides. The latter represented
genes involved in neuromuscular physiology and related diseases. They were
selected from suppression subtractive hybridization libraries, pangenomic
microarray hybridization data and literature databases. Two replicates of each
experiment were done using different microarray slides. Two statistical analysis
tools specifically dedicated to microarrays data (SAM, Significance Analysis of
Microarray and Limma, LInear Models for MicroArray data) were used. Only genes
commonly yielded by both methodologies were admitted in the final list of genes
differentially expressed in DMD biopsies compared with unaffected biopsies. RESULTS:
1009 genes were differentially expressed in dystrophic muscles as in
comparison with normal skeletal muscle samples. Part of them reflected changes
in the histological pathology (e.g. extracellular matrix, muscle
structure and regeneration). Among differentially expressed genes, a few ones
were related to two specific molecular pathways that could contribute to the
pathogenic process. Genes encoding matrix metalloproteinase 2, 3 and 14 and
tissue inhibitor metalloproteinase 1 and 2 were overexpressed, suggesting a
dysregulation of tissue remodeling pathways. Genes encoding caveolin structure (caveolin
1, 2 and flotillin 1) were also overexpressed, revealing that this signaling
pathway might be substantially affected in DMD muscle fibers. CONCLUSIONS: The
present study illustrates the potential interest of microarray technology in the
comprehension of neuromuscular disorder pathophysiology. In particular, it
suggests that metalloproteinase and caveolin signaling pathways might be
affected in the DMD dystrophic process. It also highlights a large number of
genes whose involvement in the muscular dystrophy pathogenesis was unknown.
Their identification might be important in the understanding of the disease
process.