S24.003]
Gene Transfer Clinical Trial for Limb Girdle Muscular Dystrophy Type 2D (Alpha-Sarcoglycan
Deficiency)
Jerry R. Mendell, Hansell Stedman, Steven A. Moore, Valérie Allamand, Kevin P.
Campbell, Cheryl Wall, Cathy Stolle, Isabelle Periquet, Vera Novak, James W.
Wilson Columbus , OH; Philadelphia, PA; Iowa City , IA
OBJECTIVE: Determine the safety of gene transfer in patients with limb girdle
muscular dystrophy (LGMD) type 2D (alpha-sarcoglycan deficiency)
BACKGROUND: Current treatment opportunities for muscular dystrophy are limited.
LGMD caused by sarcoglycan deficiencies are particularly suitable for
viral-mediated gene transfer to skeletal muscle using adeno-associated virus (AAV).
This replication defective, nonpathogenic vector combines high-efficiency with
low immunogenicity and can transfer the entire cDNA of the sarcoglycan genes.
AAV has been used to successfully transfer the sarcoglycan and dystrophin coding
sequences in animals.
DESIGN/METHODS: Two subjects participated in this trial, a 37-year-old man (patient
1) and a 14-year-old boy (patient 2). Both had missense mutations of the
alpha-sarcoglycan (a-SG) gene. Expression of a-SG in quadriceps muscle biopsies
prior to gene transfer was greatly reduced in patient 1 and absent in patient 2.
The full length a-SG cDNA was cloned into an AAV-2 vector plasmid containing a
cytomegalovirus (CMV) promoter to generate the vector construct, AAV-CMV-a-SG.
This construct was shown to express a-SG in a mouse model of LGMD 2D. The
extensor digitorum brevis (EDB) was chosen as the site of gene transfer and
following a double-blind, randomized protocol, one side received vector and the
other side saline. Forty-three days after gene transfer the muscles were
biopsied for analysis.
RESULTS: Both patients tolerated the injections into the EDB without adverse
effects. Neither developed signs of local or systemic reaction at any time. No
differences could be discerned in histopathology comparing the right and left
EDBs of either patient. Dystrophic changes were observed on both sides. Although
macrophages infiltrated muscle fibers undergoing necrosis, no endomysial or
perimysial inflammatory cell infiltrates were observed. Results of gene
expression will be shown at the time of presentation.
CONCLUSIONS: Several important lessons were learned from this first gene
transfer to skeletal muscle in muscular dystrophy. The patients experienced no
adverse reactions and there were no signs of inflammation detected in the muscle
biopsies post gene transfer. This sets the stage for future studies using AAV in
clinical gene transfer trials in musuclar dystrophy. The EDB posed problems in
interpretation related to retained sarcoglycan expression in these patients with
missense mutations of the a- SG gene. In future gene transfer trials limb
muscles may improve opportunities for assessing efficacy both in regard to gene
expression and for measurement of strength using quantitative methods.
Supported By: Muscular Dystrophy Association
Richard I. Webster, Alison Boynton, Richard L. Henry, Heather M. Johnston Randwick,
New South Wales, Australia
OBJECTIVE: To evaluate the use of Peak Expiratory Flow (PEF) as a measure of
muscle strength in young boys with Duchenne Muscular Dystrophy (DMD) and to
compare the use of PEF with other measures of respiratory function.
BACKGROUND: There is a need to measure the effect of treatments that potentially
increase muscle strength in DMD. Assessment of muscle strength in young boys
with DMD is often hampered by problems with attention and concentration. Vital
Capacity (VC) is the most commonly used measure of respiratory function but
young boys find it a difficult manoeuvre. PEF is easily performed, correlates
with other measures of respiratory muscle strength and so potentially has some
advantages as a measure of muscle strength.
DESIGN/METHODS: 17 boys with DMD (5-10 years of age) were seen at 4 monthly
intervals over one year. Each assessment involved respiratory function testing (PEF,
VC, one second Forced Expiratory Volume [FEV1]), timed functional testing (walking
9 metres, climbing 4 stairs and arising from supine) and manual muscle strength
testing (MMST). A single investigator performed MMST and timed functional
testing. A separate investigator performed respiratory function testing. Both
investigators were blinded to the results of the other's assessment. During the
study 11/17 boys were treated with prednisolone, which increases muscle strength
in DMD.
RESULTS: At baseline, mean PEF (69% of predicted) and VC (76% of predicted) were
abnormal. PEF and VC manoeuvres were assessed as being adequately performed in
80% and 65% of assessments respectively. Only 22% of flow-volume loops were
considered to accurately estimate VC. At baseline, there was a significant
correlation between PEF (% predicted) and strength assessed by MMST (P=0.002)
and between PEF (% predicted) and the time to walk 9 metres (P=0.01). At
baseline, neither VC (% predicted) nor FEV1 (% predicted) showed a statistically
significant correlation with MMST or timed functional testing. At the time of
the final assessment, change from baseline was calculated for each boy. There
was a significant correlation between the change in PEF (% predicted) and the
change in: MMST (P=0.03), time to climb 4 stairs (P=0.001), time to walk 9
metres (P=0.001) and time to arise from supine (P=0.002). Neither change in VC
(% predicted) nor FEV1 (% predicted) showed a statistically significant
correlation with these parameters. Boys treated with prednisolone did better
than those not treated, in all parameters. PEF, time to walk 9 metres and time
to climb 4 stairs showed the only statistically significant improvement. Mean
PEF (% predicted) showed a 20% improvement from baseline in the treated boys and
a 2% improvement in the untreated boys (P=0.007).
CONCLUSIONS: PEF is abnormal early in the course of DMD, correlates well with
other measures of muscle strength and is sensitive to changes in muscle strength
induced by prednisolone. It is more easily performed than spirometry and hence
has some advantages as a means of monitoring muscle strength in young boys with
DMD.
Supported By: The Muscular Dystrophy Association of New South Wales
P04.147]
Integrin alfa7beta1 in Muscular Dystrophy/Myopathy of Unknown Etiology
Elena Pegoraro, Paola Prandini, Marina Fanin, Guido Tarone, Eva Engvall, Corrado
Angelini Padova, Italy; Torino, Italy; La Jolla, CA
OBJECTIVE: To investigate the role of integrin alfa7 in muscle pathology we
studied a large series of muscle biopsy from patients affected with unclassified
muscular dystrophy/myopathy.
BACKGROUND: Integrins are transmembrane heterodimer glycoproteins resulting from
the assembly of two different subunits, a and b, and have a prominent role in
myogenesis, differentiation, cell migration and cell-cell interactions. The alfa7
subunit is mainly expressed in skeletal and cardiac muscle and it has been
previously shown to be primarily mutated in three patients affected with
congenital myopathy with variable clinical phenotype.
DESIGN/METHODS: We used a candidate gene and protein approach in a large cohort
of muscular dystrophy/myopathy patients. Antibody against the intracellular
domain of the integrin alfa7 was used to immunostain muscle biopsy from 210
patients with muscular dystrophy/myopathy of unknown etiology. Patients whose
muscle biopsy specimen showed integrin a7 deficiency on immunostaining were
studied for integrin alfa7 gene mutations with reverse transcription of muscle
RNA analysis involving single strand conformational polymorphism and sequencing.
RESULTS: Levels of alfa7 integrin were found to be decreased on immunostaining
of muscle biopsy specimen from 35 of the 210 patients (~17%). In six of these
patients no integrin a7 was detected. Screening for alfa7 mutation in 30 of the
35 patients detected only one integrin alfa7 missense mutation (the mutation on
the second allele was not found) in a patient presenting with a congenital
muscular dystrophy (CMD)-like phenotype (floppy infant with multiple joint
contractures and abnormal white matter signal at brain CT scan). No integrin alfa7
gene mutations were identified in all the other patients showing integrin alfa7
deficiency. Moreover, we identified a novel integrin alfa7 isoform presenting 71
bp deletion. This isoform results from a partial deletion of exon 21 due to the
use of a cryptic splice site generated by a G to A missense mutation at
nucleotide position 2644 in integrin alfa7 cDNA. This spliced isoform is present
in about 12% of the chromosomes studied.
CONCLUSIONS: Our results suggest that secondary integrin alfa7 deficiency is
rather common in muscular dystrophy/myopathy of unknown etiology. This is not
surprising considering the central role played by alfa7beta1 integrin in
anchoring the intracellular cytosketon via actin, to the extracellular matrix,
via laminin, and the potential for signal transduction possibly mediated by
extracellular matrix ,soluble growth factors, and/or associated transmembrane
molecules. Thus, several distinct primary defects may converge into an integrin
common pathway system determining underexpression of integrin as a modulatory
phenomenon.
Supported By: Telethon grant # 1114
S24.004]
Novel Sarcoglycan Mutations Widen the Clinical Spectrum of Limb-Girdle Muscular
Dystrophy 2C,2D,2E,2F
Corrado I. Angelini, Chiara Boito, Marina Fanin, Gabriele Siciliano, Elena
Pegoraro Padova, Italy; Pisa, Italy
OBJECTIVE: To identify primary sarcoglycan mutations in a group of 221 autosomal
recessive limb-girdle muscular dystrophy (LGMD) and myopathy patients.
BACKGROUND: A new clinical molecular classification of Limb-girdle muscular
dystrophy has been proposed: the defect of four transmember glycoproteins alpha,
beta, gamma, delta sarcoglycan results in LGMD type 2D, 2D, 2E, 2C and 2F
respectively. All sarcoglycan defects cause the loss of SG (sarcoglycan) complex
and are due to very heterogeneous set of mutations that cause different
molecular consequences. The loss of SG complex leads to membrane instability in
skeletal and cardiac muscle. A possible mechanism has been identified since SG
complex deficiency leads to the unmasking of metallo-proteases cleavage site on
beta-dystroglycan, causing the disruption of the link between the basement of
membrane and sarcolemma.
DESIGN/METHODS: Muscle biopsy of 221 LGMD/myopathy patients were analized by
immunohistochemistry and immunoblot to study the expression of four subunits of
SG complex (alpha,beta, gamma, delta SG). Abnormal SG expression was found in 24
cases. RNA was extracted from biopsies and was studied by RT-PCR and SSCP
analysis, we identified the mutations by cDNA direct sequencing.
RESULTS: We found 7 patients with novel frameshift and missense mutations in
alpha-SG: their clinical phenotype ranged from hyperCKemia to mild myopathy or
severe LGMD. One patient showed an homozygous out of frame duplication of 32
base pair in beta-sarcoglycan gene and presented a severe dilated cardiomyopathy,
3 patients had mutation in gamma-sarcoglycan and presented severe muscular
dystrophy. We found a novel delta-SG mutation in a female with a limb-girdle
phenotype.
CONCLUSIONS: Our data combined with our previous screening study suggest that in
our patients population sarcoglycanopathy account for about 15% of LGMD cases.
The most frequent mutations were in alpha-SG which result in a spectrum of
clinically heterogeneous phenotypes ranging from a rapid progressive to a late
onset slow course. We describe a new further delta-sarcoglycan mutation causing
a mild LGMD phenotype. In our study the more severe clinical phenotypes were in
LGMD 2E and 2D. Dilated cardiomyopathy was prominent in beta-SG patient and
widens the clinical phenotype.This might be explained by the involvement of
vascular smooth muscle, were beta-sarcoglycan is present, leading to coronary
disfunction and myocardial necrosis.
Supported By: Telethon project n.1321-1114,C.54.
Renald Gilbert, An-Bang Liu, Basil J. Petrof, Josephine Nalbantoglu, George
Karpati Montreal , QC, Canada; Hualien, Taiwan
OBJECTIVE: To construct a fully gutted adenovirus (Ad), also known as
helper-dependent Ad (HDAd), for efficient and long-term expression of dystrophin
in muscle.
BACKGROUND: HDAd hold great promises as a gene transfer vector for the treatment
of genetic diseases such a Duchenne muscular dystrophy. They do not encode any
viral genes and are consequently less immunogenic than E1-deleted Ad (first
generation Ad). Moreover, HDAd have an increased transport capacity and can
carry up to 35 kb of foreign DNA. In addition to the vector, the intensity and
duration of the transferred gene expression is greatly influenced by the
promoter. Therefore we investigated a hybrid promoter (cytomegalovirus enhancer/b-actin
promoter), known as CB, whose efficiency is expected to be superior to any
previous promoters employed in the context of a HDAd. To further increase the
amount of transgene protein produced, two tandem dystrophin expression
cassettes regulated by the CB promoter were inserted into the backbone of the
HDAd.
DESIGN/METHODS: A HdAd without any viral genes and encoding two human
full-length dystrophin cDNAs (12 kb each) regulated by the CB promoter was
constructed and called HDCBDysDys. This vector was amplified by several passages
on 293cre cells and purified by centrifugation on CsCl gradients. The tibialis
anterior (TA) muscle of neonatal and young adult dystrophin-deficient mdx mice
was injected with 5 ml or 30 ml respectively of HDCBDysDys at a titer of 1.0 X
1012
virus particles/ml. At 10 and 30 days (adults) or at 10, 30 and 90 days
post-injection (neonatals), the injected muscles were analyzed by
immunohistochemistry for dystrophin expression. The sera of some animals were
also analyzed by western blot for the presence of antibodies against dystrophin.
RESULTS: At 10 days post-injection, the average number of dystrophin positive
fibers/muscle was 582 (34% of total) and 104 (6.1% of total) for neonatal and
adult mdx mice respectively. In addition, many transduced fibers demonstrated an
intense dystrophin positive cytoplasmic staining, a sign of marked dystrophin
over-expression. This transduction level was significantly higher than the one
observed after gene transfer with a HDAd encoding a single dystrophin expression
cassette regulated by the cytomegalovirus promoter (Gilbert et al., Hum. Gene
Ther. 12:1741). However, significant reduction in the number of dystrophin
positive fibers was observed after 30 and 90 days in the two animal groups.
Antibodies against the human dystrophin transgene were demonstrated in the sera
of some animals, indicating the transgene protein was immunogenic.
CONCLUSIONS: Very high but transient dystrophin expression was observed after
gene transfer with HDCBDysDys. It remains unclear if the progressive reduction
of the transgene expression was due to the antigenicity of dystrophin or to
other factors unrelated to the immune response. These questions are being
investigated by repeating the previous experiments in immunodeficient mice and
by generating a HDAd encoding the murine dystrophin.
Supported By: Muscular dystrophy association of USA and Canada
Renald Gilbert, Jatinderpal R. Deol, An-Bang Liu, Joon-Shik Moon, Basil J.
Petrof, Josephine Nalbantoglu, George Karpati Montreal , QC, Canada;
Hualien, Taiwan
OBJECTIVE: To test if retention of two adenoviral genes i.e. E1B and E4 in the
backbone of an otherwise gutted adenovirus (Ad) will produce and efficient and
prolonged transgene expression.
BACKGROUND: Helper-dependent Ad (HDAd), also known as fully gutted Ad hold great
promises as gene transfer vector for treating genetic diseases such as Duchenne
muscular dystrophy. HDAd are more advantageous than E1-deleted Ad (first
generation) because they encode either no or few viral genes and are
consequently less immunogenic. However, in comparison to first generation Ad,
the level of transgene expression was significantly weaker after gene transfer
with a fully deleted HDAd in muscle even if the transgenes were regulated by the
same cytomegalovirus (CMV) promoter (Gilbert et al., Hum. Gene Ther. 12:17411).
In an attempt to improve the transgene expression, we have tested a partially
gutted HDAd (AdRP1001) that encodes the E1B and E4 regions of Ad genome and
b-galactosidase (b-gal) as transgene controlled by the CMV promoter.
DESIGN/METHODS: AdRP1001 was propagated on 293Cre cells and purified by
centrifugation on CsCl gradients. The tibialis anterior (TA) muscles of
neonatal, dystrophin-deficient mdx mice, Cd1 and immunodeficient scid mice were
injected with 5 ml of AdRP1001 at a titer of 1.0 X 1012
virus/ml and the number of muscle fibers expressing b-gal was analyzed at 10,
30, 90 and 180 days later by histochemistry. The TA of neonatal mdx mice was
also injected with 5 ml or HDCBDys alone or in the presence of AdRP1001, both
vectors being injected at the same titer (5.0 X 1011
virus/ml). The number of dystrophin positive fibers was then evaluated 10 days
later by immunohistochemistry. HDCBDys is a HDAd encoding the full-length
dystrophin regulated by the strong hybrid CMV enhancer/b-actin promoter (CB)
promoter.
RESULTS: At 10 days post-injection, the average number of b-gal positive fibers
was 792 (47% of total), 1113 (65% of total) and 820 (48% of total) in mdx, Cd1
and scid muscles respectively. Such a high transduction level has not been
achieved previously by us using a fully deleted HDAd encoding dystrophin
regulated by the CMV promoter (Gilbert et al., Hum. Gene Ther. 12;1741).
Furthermore, no reduction in the number of transduced fibers was observed at 30,
90 and 180 days post-injection in scid mice. In mdx muscles, an average of 38
dystrophin positive fibers was observed after gene transfer with HDCBDys alone.
This number increased to 378 in the presence of AdRP1001.
CONCLUSIONS: Ad gene products (E1B and/or E4) encoded by AdRP1001 can
dramatically improve the expression of a transgene carried by a gutted Ad in
muscle when the transgene is controlled either by the CMV or the CB promoter.
More importantly, this elevated early transduction level remained unabated for
at least 180 days in immunodeficient animals. The inclusion of E1B and/or E4 in
a gutted Ad encoding dystrophin will most likely help in obtaining elevated and
prolonged dystrophin expression in dystrophin-deficient muscle.
Supported By: Muscular dystrophy association of USA and Canada
[P01.064]
Therapeutic Gene Replacement to Skeletal Muscle Made Efficient without Viral
Vectors
George Karpati, Renald Gilbert, An-Bang Liu, Joon-Shik Moon, Basil J. Petrof,
Josephine Nalbantoglu Montreal, QC, Canada; Hualien, Taiwan; Monteal, QC,
Canada
OBJECTIVE: To evaluate if intramuscular injection of naked DNA (plasmid),
followed by percutaneous application of electric current (electroporation, EP)
is a potentially useful strategy for treating genetic diseases such as Duchenne
muscular dystrophy.
BACKGROUND: Plasmid is an alternative to viral vectors for the transfer of
therapeutic genes into muscle. Plasmids are easier to produce, safer and cheaper
then viral vectors. However, transfection level after intramuscular injection of
plasmid is very low. EP has been demonstrated to greatly improve the
transfection level after plasmid injection into muscle. Pre-treatment of muscle
with hyaluronidase (Hy) increases the transfection level after EP. In the
present study, we have investigated the effects of age and strain of animals on
EP and Hy. Two age groups and three strains of mice were studied after
intramuscular injection of plasmids.
DESIGN/METHODS: Three plasmids were used: pCBLacZ and pCBMuDys encoding b-gal
and murine dystrophin respectively, both transgenes being controlled by the
hybrid cytomegalovirus (CMV) enchancer/b-actin promoter (CB promoter); plasmid
pCMVUtrFl encoding murine utrophin regulated by the CMV promoter and tagged with
a Flag epitope. The TA of 5-week-old Cd1, C57BL/6 and dystrophin-deficient mdx
mice, as well as 15-day-old mdx mice were injected with 1 mg/ml of plasmid
followed by 8 pulses (duration: 20 msec; interval: 1 sec) of 175-200 V/cm. Some
TA were also injected with Hy (0.4m/ml) two hours before EP. At 6 to 8 days
post-injection, the transfection level was determined by processing the muscles
for histochemistry and immunohistochemistry.
RESULTS: In adult mdx, C57Bl/6 and Cd1 animals, plasmid alone produced 3-10
transfected fibers. In the same model, EP markedly increased the transfection
level of b-gal expressing plasmid in TA muscle to 169 (10%), 279 (16%) and 326
(19%) fibers respectively. EP+Hy further increased the transfection level by 2-3
folds. Although a trend was observed toward lower transduction level in mdx mice,
the difference was not significant. In very young mdx animals, b-gal expressing
plasmid plus EP produced very low transfection (5 fibers/muscle). EP+Hy produced
120 fibers/muscle. Adult mdx muscles injected with plasmid expressing either
murine dystrophin or utrophin plus EP+Hy produced up to 144 and 276 transfected
fibers/muscle respectively.
CONCLUSIONS: Pre-treatment with Hy followed by EP markedly increase the
transduction level after intramuscular injection of plasmid DNA in adult normal
(Cd1 and C57Bl/6) and dystrophic muscles (mdx). The relatively high level of
dystrophin and utrophin gene transfer following Hy and EP treatments will permit
to study in dystrophic muscles many aspects of dystrophin vs utrophin gene
transfer without interference by viral vectors.
Supported By: Muscular dystrophy association of USA and Canada.
P01.067]
Novel Dystrophin Gene Mutations and Unusual DMD/BMD Phenotypes
Elicia Estrella, Benjamin Roa, Thomas Prior, Jerry Mendell, Lisa Baumbach Miami,
FL; Houston, TX; Columbus , OH
OBJECTIVE: This study illustrates several unusual clinical presentations of DMD/BMD.
BACKGROUND: Since the discovery of the Duchenne Muscular Dystrophy (DMD) gene
approximately 15 years ago, hundreds of Duchenne, Becker, and other patients
with a suspected primary dystrophinopathy have been evaluated for mutations in
the DMD/BMD locus. Knowledge gained from these studies has led to general
genotype: phenotype predictions, although these predictions do not nearly
account for the spectrum of clinical variability noted. The detection of
patients with novel mutations may provide new insights into functional aspects
of the dystrophin gene and/or associations with unusual clinical features.
DESIGN/METHODS: Patients were evaluated for deletions/ duplications in the
dystrophin gene. Molecular analyses were completed via standard PCR and southern
blotting techniques. In several patients, Western blot analysis was performed
for quantitative and qualitative studies.
RESULTS: We report our investigations of several unusual DMD/BMD patients. The
first two patients displayed no previous family history of muscle disease and/or
similar symptoms. Patient 1, an 11 1/2 year old Caucasian male, was referred for
a long undefined history of weakness and unsteadiness. He had moderate serum CK
elevations. At time of examination, he had no pseudohypertrophy, no significant
weakness, a modified Gowers maneuver, and normal reflexes in all extremities.
His only clinical complaint was of pain (cramps) after strenuous exercise.
Mutation studies of the DMD gene revealed a deletion spanning exons 3-4. This
deletion has been reported in three other Beckers patients (Leiden Muscular
Dystrophy mutation database). Although the deletion is found in the beginning of
the gene, and presumably affects mRNA splicing, it is very interesting that all
patients exhibit an extremely mild Beckers phenotype. Patient 2, a 6 1/2 year
old African-American male, displays marked developmental delay for all
milestones (motor and cognitive) since infancy. He also has speech delay, and
ADHD. These features are not present in any other family members. Clinical exam
noted minimal muscle involvement including upper extremity weakness (since age
3), mild calf pseudohypertrophy, no ambulatory problems, but markedly elevated
serum CK levels. DNA studies revealed a possible duplication of exons 10-12 and
the African-American exon 8/9 RFLP. Review of the literature indicates that this
is a novel mutation, associated with a gross gene rearrangement. His phenotype
is unusual for the marked level of mental impairment. Patients three-five are
all classical BMD patients who share a common exon 45-53 in-frame deletion.
Western blot analysis of muscle dystrophin revealed a normal level (100%) of the
truncated, mutant form. DMD/BMD deletions which produce quantitatively normal
dystrophin levels should be further investigated.
CONCLUSIONS: It is our hope that similar patient descriptions will lead to
further understanding of the dystrophin-associated muscular dystrophies. Insight
gained from these genotype:phenotype correlations may lead to better progostic
and treatment capabilities.
[P04.145]
Relatively Prevalent Mutations in the Dysferlin Gene and Genotype-Phenotype
Correlation of Miyoshi Myopathy in Japanese Population
Toshiaki Takahashi, Masashi Aoki, Maki Tatayama, Yoshiaki Onodera, Yuji Hinuma,
Emi Kondo, Ikuya Nonaka, Kiichi Arahata, Robert H. Brown, Jr., Hiroshi Saito,
Yasuto Itoyama Sendai, Miyagi, Japan; Kodaira, Tokyo, Japan; Charlestown,
MA
OBJECTIVE: To reveal the features of dysferlin gene mutations in Japanese
patients with Miyoshi myopathy (MM) and a genotype-phenotype correlation in the
disease.
BACKGROUND: MM is an autosomal recessive distal muscular dystrophy. The gene
dysferlin was identified recently and found to be mutated both in families with
MM and those with limb girdle muscular dystrophy type 2B.
DESIGN/METHODS: We examined 26 Japanese patients with MM. Genomic DNA was
extracted from the peripheral lymphocytes of the patients with informed consent.
The PCR products of each 55 exon were screened by single strand conformation
polymorphism (SSCP) or direct sequencing from the PCR fragments.
RESULTS: We identified 16 different mutations in 20 MM patients and 10 of them
were novel. Mutations in Japanese patients are distributed along the entire
length of the gene. The mean age at onset of the patients with the dysferlin
mutations was 23.7 ± 7.3 years (from 14 years to 37 years). The mean serum CK
level of the patients was 5,326 ± 3,152 IU/l (min 1,289 IU/l, max 12,556 IU/l).
Residence of the patients with the dysferlin mutations had a wide distribution
in Japan.
CONCLUSIONS: We found relatively prevalent four mutations (C1939G, G3370T,
3746delG, and 4870delT) in these populations, and they accounted for 60 percent
of the mutations in this study. We speculated that most patients with MM were
selectively affected in the paravertebral muscles even in the very early stage.
This study revealed that the G3370T mutation may be associated with the mild
form of MM whereas G3510A mutation may be associated with the severe form of MM.
Supported By: Grant-in-Aid from the Ministry of Health, Labor and Welfare in
Japan and Grant-in-Aid for Scientific Research from Japan Society for the
Promotion of Science.
P01.063]
Myogenesis of Human Adult Dermal Fibroblasts (HADFs) In Vitro:: Potential Source
for Gene Therapy of Muscular Dystrophies
Tokuro Furuya, Hiroaki Tanaka, Noriyoshi Kameda, Fumiko Saito, Takayoshi
Kobayashi, Hidehiro Mizusawa Bunkyo-ku, Tokyo, Japan; Nakano-ku, Tokyo,
Japan
OBJECTIVE: To clarify whether HADFs particitate in the myotube formation.
BACKGROUND: Skeletal muscles are generated mainly from myogenic precursor cells.
However, recently some reports have indicated that other type of cells could
become the source of myogenesis such as bone marrow.
DESIGN/METHODS: HADFs from human skin biopsied with informed consents were
cultured in several passages and labeled with marker gene LacZ encoding
bacterial beta-galactosidase. Same numbers of labelled HADFs and mouse myoblasts,
C2C12 cells, were co-cultured. To identify the origin of the nuclei of myotubes
in co-culture, we performed Fluorescence in situ hybridization (FISH) using two
probe which contain human or mouse specific chromosome repetitive sequence. To
investigate whether HADFs-nuclei in myotubes expressed muscle specific
transcripts, RT-PCR primers were designed in the 3' UTR region of human myogenin
gene. HADFS with marker gene were co-cultured with human muscle cells innervated
with fetal rat spinal cords whether they particitate mature innervated muscle
fibers.
RESULTS: When HADFs were co-cultured with mouse myoblasts, beta-galactosidase
positive myotubes appeared, which were supposed to have HADFs-derived nuclei.
FISH revealed that these myotubes have at least one human nuclei among their
multiple nuclei. In co-culture of HADFs and mouse myoblasts, human specific
myogenin transcripts were detected by RT-PCR. Furthermore when LacZ introduced
HADFs were co-cultured with human muscle cells innervated with fetal rat spinal
cord, beta-galactosidase positive striated contracting mature muscle fibers were
formed.
CONCLUSIONS: This is the first report to clarify that HADFs have the potential
capacity of participating myogenesis in vitro. HADFs may become the alternative
source of cell transplantation therapy for muscular dystrophies
Shree Pandya, Gary J. Myers, Richard T. Moxley Rocheste, NY; Rochester, NY
OBJECTIVE: To document the effect of long term prednisone treatment on
ambulation in patients with Duchenne dystrophy.
BACKGROUND: Three double blind, randomized, controlled trials and a three year
follow up open trial have clearly documented that prednisone increases muscle
strength and muscle mass, and slows progression of disease. Prednisone
administered daily at a dose of 0.75mg/kg/d increases strength over a three
month period, stabilizes it for eighteen months or more, and slows progression
of the disease over at least a three year period. DeSilva et al in a
retrospective study of 16 patients found that a subset of patients who received
prednisone for 2 years or more, lost independent ambulation at an average age of
13.1, compared to the control group who lost independent ambulation at 10.3, a
gain of almost 3 years.
DESIGN/METHODS: We have prospectively followed a group of patients with Duchenne
dystrophy who began prednisone treatment between 1986 and 1989 as part of the
Clinical Investigation of Duchenne Dystrophy (CIDD) therapeutic trials at our
center. After completion of the trials, these patients have received 0.75mg/kg/d
of prednisone unless side effects required a reduction in dosage. Patients have
returned twice yearly for evaluations which included measurements of vital signs,
height, weight, timed function tests and side effects.
RESULTS: We have followed 30 patients for an average of 11.2 years (range 3-15
yrs). At the initiation of prednisone, 18 of the 30 were ambulatory. 13 walked
independently (avg age 8.3 yrs) and 5 walked with long leg braces (avg age 10
yrs). At last contact all patients had become wheelchair dependent. One patient
has been lost to follow up. Three patients discontinued Prednisone due to weight
gain. The average age at loss of independent ambulation is 14.6 years (range 12
to 19 yrs).
CONCLUSIONS: Loss of independent ambulation has been reported to occur between
the ages of 8.5 and 10.8 years in untreated patients. In our series, patients
treated with prednisone have ambulated independently until age 14.6, a gain of 4
to 6 years compared to untreated controls reported in the literature. Long term
treatment with prednisone substantially lengthens independent ambulation in
patients with Duchenne dystrophy.
Thomas A. Rando, Carmen Bertoni Stanford, CA
OBJECTIVE: To test for chimeraplast-mediated repair of the dystrophin gene point
mutation in mdx muscle precursor cells.
BACKGROUND: Chimeraplasts are chimeric oligonucleotides made up of both DNA and
RNA bases that have been found to be effective as gene therapy agents to correct
point mutations in genomic DNA. They are designed to be homologous to a region
of DNA with a single mismatch at the base targeted for correction. The mechanism
of repair involves first homologous pairing followed by the induction of
cellular mismatch repair activities. Chimeraplasts have been shown to induce
repair of point mutations in different cell types, both in vivo and in vitro. We
have previously demonstrated that injection of MDX1, a chimeraplast targeted to
correct the point mutation in the mdx dystrophin gene, into muscles of mdx mice
leads to repair of that mutation and to dystrophin protein expression in
differentiated myofibers in vivo. We wanted to determine whether MDX1 would also
correct the point mutation in mdx muscle precursor cells (satellite cells or
myoblasts) in order to further assess the potential for chimeraplasts to be
developed for in vivo or ex vivo gene therapy.
DESIGN/METHODS: Myoblasts were derived from mdx muscle and propagated in vitro.
MDX1 or control (MDX2) chimeraplasts were transfected into those cells. After
growth in culture, repair of the mdx point mutation was assessed at the genomic
level using the mdx-ARMS assay. Alternatively, cells were induced to
differentiate into myotubes, and correction of the mutation was assessed at the
mRNA level by RT-PCR-ARMS assay. Differentiated cultures were also assessed for
dystrophin protein expression using both Western analysis and
immunohistochemical staining. For in vivo experiments, chimeraplasts were
directly injected into muscles of mdx mice. Two days later, primary cultures
were established from those injected muscles and the myoblasts in the cultures
were assessed for dystrophin gene correction by the same methodologies.
Wild-type (C57) myoblasts and mice were used as controls.
RESULTS: Correction of the mdx point mutation by MDX1, but not MDX2, was
demonstrated at the genomic level in muscle precursor cells after in vitro
transfection or in vivo injection. Similarly, transcripts containing the
wild-type dystrophin sequence were demonstrated in mdx myotube cultures in both
experimental paradigms. Immunohistochemical analysis of differentiated cultures
demonstrated dystrophin protein expression in multinucleated myotubes. Western
analysis demonstrated that the level of dystrophin expression in MDX1-transfected
cells, 4 weeks after transfection and 3 days after differentiation, ranged from
2% to 15% (n=6) of that seen in C57 myotube cultures.
CONCLUSIONS: Chimeraplasts can effect gene repair in muscle precursor cells,
both in vitro and in vivo. This is a promising finding for using chimeraplasts
for either in vivo or ex vivo gene therapy for dystrophies due to point
mutations of the dystrophin gene. Further studies are focusing on methods to
improve the efficiency of gene repair still further and establishing methods for
in vivo delivery of chimeraplasts.
Supported By: Study supported by a grant from the Muscular Dystrophy Association
to TAR.
Romain K. Gherardi, Corinne Sonnet, Peggy Lafuste, Benedicte Chazaud Creteil,
France, Metropolitan
OBJECTIVE: To characterize the interplay between myogenic precursor cells (mpc)
and monocyte/macrophages (m/M).
BACKGROUND: Cell therapy using myoblast transfer is based on capacity of mpc to
be activated after muscle injury, to proliferate and to fuse into new muscle
fibers. To develop their myogenic program, mpc depend from environmental cues,
the so-called biologic niche. Macrophages are crucially involved in skeletal
muscle regeneration: after injury, the muscle fiber is invaded by macrophages
and regeneration is markedly impaired in the absence of macrophage infiltration.
In addition to muscle cell debris removal by phagocytosis, macrophages likely
participate to the subsequent phases of muscle reconstruction. Investigating
this topic should allow optimisation of future cell therapy programs.
DESIGN/METHODS: Mpc:m/M interplay was assessed using human cells according to a
3-step methodology: 1) assessment of elementary biologic effects on cell density,
phagocytosis, and chemotaxis through porous filters or endothelial cells; 2)
selection of candidate effector molecules for the observed effects using a DNA
array procedure; 3) validation of the role of selected candidate molecules by
specific inhibition tests.
RESULTS: Mpc exerted a chemotactic effect toward monocytes, which effect was
potentiated by 35% when mpc were previously stimulated by macrophages.
Macrophages alone exerted a weaker chemotactic effect toward monocytes than mpc
alone, but stimulation of macrophages by mpc increased the chemotactic activity
of macrophages by 80%.
DNA array displayed expression of three chemokines by mpc that could be
responsible for chemotaxis: MCP-1 (CCL2), MDC (CCL22) and fractalkine (CX3CL1).
Monocyte chemotaxis toward mpc was inhibited from 40 to 60% in the presence of
blocking antibodies against these chemokines. The 3 antibodies mixed together
reduced chemotaxis by 90%.
Mpc:M cocultures showed no phagocytosis of living mpc. Moreover, macrophages
increased mpc density in a dose-dependent manner, a 1:2 (mpc:M) ratio inducing a
85% increase of mpc density.
CONCLUSIONS: Activated human mpc recruit circulating monocytes and stimulate the
recruited macrophage chemotactic activity. In turn, macrophages increase mpc
chemotactic activity and boost mpc growth.
Supported By: Association Francaise contre les Myopathies (AFM)
Ami Mankodi, Christina Jiang, Masanori P. Takahashi, Carol Beck, Richard T.
Moxley, Stephen C. Cannon, Charles A. Thornton Rochester, NY; Boston, MA;
Philadelphia, PA; Boston, MA
OBJECTIVE: Determine the cause of myotonia in DM type 1 (DM1) and DM type 2
(DM2).
BACKGROUND: DM is caused by expansion of a CTG repeat on chromosome 19 (DM1) or
a CCTG repeat on chromosome 3 (DM2). Manifestations of DM result from
accumulation in the nucleus of RNA with expanded CUG repeats (DM1) or CCUG
repeats (DM2). Transgenic mice that express expanded CUG repeats (HSA-LR mice)
also develop myotonia and a DM-like myopathy. While myotonia implies dysfunction
of ion channels, the nature of the channelopathy in DM is unknown.
DESIGN/METHODS: Ion conductance in the transgenic model was assessed by making
intracellular recordings from muscle fibers of HSA-LR or wild-type (WT) mice.
Expression of the chloride channel 1 (ClC-1) mRNA in murine and human DM was
assessed by Northern blot, real-time RT-PCR, and sequence analysis of cDNA
clones to evaluate RNA splicing. Distribution of Cl channel was assessed by
immunofluorescence (IF) using polyclonal antibody to the C-terminus of ClC-1.
RESULTS: Compared to WT mice, Cl conductance in HSA-LR mice was reduced 10-fold,
an alteration sufficient to cause myotonia. To investigate the mechanism for
reduced chloride conductance, we examined the expression of ClC-1, the
predominant Cl channel in muscle. HSA-LR mice showed a major defect in splicing
of the ClC-1 pre-mRNA. Among 29 ClC-1 cDNA clones, only 24% encoded full-length
protein. 76% of clones were incorrectly spliced; nearly all had premature
termination codons. A wide range of splicing errors was observed (11 different
anomalous splice sites). The total level of ClC-1 mRNA in HSA-LR muscle was
slightly reduced (~40% of WT), presumably due to accelerated degradation, via
the nonsense-mediated decay pathway, of mis-spliced mRNA. Results in human DM
were similar: the fraction of aberrantly-spliced ClC-1 clones was >95% in
severely-affected patients with DM1 (n=2), and > 70% in patients with DM2
(n=2). IF on muscle sections from HSA-LR mice and from patients with DM1 or DM2
revealed a mosaic pattern in which some muscle fibers had a normal
circumferential rim of ClC-1 protein, but most were devoid of ClC-1 or had
interruptions in the sarcolemmal rim of staining.
CONCLUSIONS: Our results support a model in which nuclear accumulation of RNA
with expanded CUG or CCUG repeats causes aberrant splicing of ClC-1 mRNA, loss
of ClC-1 protein, reduced chloride conductance, and consequently
hyperexcitability of the muscle membrane in both DM1 and DM2.
Supported By: Supported by NIH and the Muscular Dystrophy Association.
Eduard Gallardo, Mengfatt Ho, De Luna Noemi, Diane Mckenna-Yasek, Robert H.
Brown, Isabel Illa Barcelona, Spain; Boston, MA
OBJECTIVE: To develop a new dagnostic assay for limb girdle muscle dystrophy 2B
(LGMD2B), Miyoshi myopathy (MM)and distal anterior compartment myopathy (DAT),
which screens for dysferlin expression in blood using a commercially available
monoclonal antibody.
BACKGROUND: Recently, LGMD2B,MM and DAT were found to be allelic disorders
arising from defects in a novel gene that encodes dysferlin. The dysferlin gene
is large comprising 55 exons and encodes a protein of 237 kDa. Dysferlin is
localized to the plasma membrane of muscle fibers. Its function is unknown,
however its homology with a gene of the nematode Caenorhabditis elegans,fer-1,
suggests that it may function in membrane fusion or trafficking. The size of the
gene and the fact that most mutations are single nucleotide changes make
molecular diagnosis difficult. Western Blot(W-B) analysis of muscle biopsies has
been shown a reliable and rapid method for the diagnosis of muscle dystrophies.
However, muscle biopsy is an invasive procedure.
DESIGN/METHODS: We isolated peripheral blood mononuclear cells (PBMC) from whole
blood of patients with disferlinopathy (n=12) and controls using a Ficoll-Plaque
gradient centrifugation. Total RNA was isolated and we performed a Northern-Blot
(N-B)with specific probes. W-B and immunocytochemistry using a monoclonal
antibody anti-dysferlin (NCL-Hamlet)were also performed. In addition, we
isolated CD14+ (a monocyte marker) cells by means of magnetic beads and column
separation, and performed W-B and immunocytochemistry using the same antibody.
RESULTS: N-B analysis of total RNA from blood showed a weak but distinct band of
about 7.5 kb, corresponding to the dysferlin transcript. A prominent band of
230kDa was detected by W-B using PBMC extracts from controls but not in patients.
Immunocytochemistry confirmed the results. Immunocytochemistry and W-B of
purified CD14+ cells verified that expression of dysferlin was restricted to
monocytes. Skeletal muscle from all patients was also tested for dysferlin
expression and it was absent in all of them.
CONCLUSIONS: Dysferlin is expressed in monocytes CD14+ and correlates with its
expression in skeletal muscle. This new blood-based diagnostic assay is a simple
and rapid technique and offers a less invasive tissue sampling compared to
muscle biopsy. The discovery of expression in monocytes provides a new paradigm
to study the biological function of this protein in a non-muscle cell type that
is more accesible. Also, for experimental therapies intended to increase the
levels of dysferlin, we could now track efficacy by monitoring dysferlin levels
in blood rather than skeletal muscle.
Supported By: FIS 99/019-01; FIS 01/0979; MDA (USA); C.B.Day Investment Company;
NIH
Christiane Schneider, Marcus Beck, Annette George, Carsten Wessig, Henrik Kele,
Wolfram Kress, Karlheinz Reiners, Klaus V. Toyka Würzburg, Germany; Göttingen,
Germany
OBJECTIVE: To investigate the efficacy and safety of creatine monohydrate (Cr)
in patients with the proximal myotonic myopathy (PROMM) phenotype.
BACKGROUND: PROMM is characterised by proximal weakness, myotonia, myalgia and
cataracts. Fluctuations of weakness, myotonia and muscle pain occur frequently
in PROMM suggesting that disturbances of muscle energy metabolism might be
involved. Cr is known to have various positive effects on muscle cell metabolism
and was shown to be efficient in muscular dystrophies and mitochondrial
myopathies.
DESIGN/METHODS: Twenty patients (10 men, 10 women) with PROMM with overt
neuromuscular weakness and myotonia were randomised to either the Cr or the
placebo group. Cr was given for 3 months in a daily dose of 10 g, i.e. 18 ml of
pure Cr powder. Controls received the same volume of microcrystalline cellulose.
muscle strength sum scores using the Medical Research Council (MRC) scale, the
Neuromuscular Symptom Score (NSS) and hand-held dynamometry, and a self
assessment analogue scale were evaluated seperately for each treatment group
before and after the treatment period by a blinded examiner. We defined changes
in average MRC and hand-held dynamometry sum scores and NSS as primary endpoints.
RESULTS: Using the strength measurement methods described above no significant
improvement of muscle strength was found in the total Cr group as compared to
the placebo group and in the male patients of both groups (Mann-Whitney U-test,
p > 0.05). In the gender subgroup analysis muscle strength improved
significantly in women who had received Cr as compared to women who had received
placebo (p = 0.005 (hand-held dynamometry) and p = 0.03 (MRC scale),
Mann-Whitney U-test). Seven patients of the Cr group and one patient of the
placebo group reported subjective improvement with increased endurance in daily
activities and sports. During the administration of Cr leg muscle pain resolved
only in one male and one female patient. Chest pain resolved in another male
patient.
CONCLUSIONS: Cr was well tolerated during the treatment period. In this pilot
study, Cr had a beneficial effect on muscle strength only in women. In addition,
Cr occasionally had a positive effect on muscle and chest pain. Larger studies
are necessary to confirm the results of this study.
Supported By: Verein der Freunde und Förderer der Neurologischen Klinik der
Universität Würzburg. Cr and placebo medication were kindly provided by SKW
Trostberg, Trostberg, Germany.
[P03.132] Cardiac Involvement
in Young Myotonic Dystrophy (DM1) Patients
Guillaume Bassez, Isabelle Desguerre, Arnaud Lazarus, Pascal Laforêt, Henri
Marc Bécane, Jean Varin, Hélène Radvanyi, Bruno Eymard, Denis Duboc Créteil,
France; Paris, France
OBJECTIVE: To determine the frequency of severe cardiac symptoms in young
patients with myotonic dystrophy (DM1).
BACKGROUND: Cardiac symptoms, one component of DM1 systemic features, have been
identified as a crucial prognostic factor in adults. Significant early cardiac
involvement in young affected individuals is usually considered as uncommon.
However, there is little available data concerning cardiac symptoms for children
and young adults. It is also unclear whether severe cardiac signs may be
encountered as an isolated inaugural symptom in DM1 adolescents, or constantly
combined with other significant clinical DM1 signs. Cardiac evaluation in young
DM1 patients (<18 year old) may be of great interest for both follow-up and
management of cardiac disorders in childhood type of DM1, and may add useful
information for genetic counselling for asymptomatic individuals at risk under
the age of 18.
DESIGN/METHODS: In a retrospective study of DM1 patients, we analysed cases with
severe cardiac involvement identified before the age of 18. Age at onset,
detailed cardiac, neuromuscular, and other systemic clinical features, results
of cardiac investigations (conduction defects, arrhythmia, ventricular
dysfunction), severity, follow-up, and number of (CTG)n triplet repeats
were investigated.
RESULTS: Six young individuals were identified with severe cardiac involvement
before the age of 18. Two subgroups can be differentiated : cardiac signs during
previously diagnosed childhood or congenital type of DM1, and severe cardiac
events inaugurating DM1 in asymptomatic teenagers. In the first group (n=3),
cardiac abnormalities were sudden death in a 17 year old boy with pace-maker,
due to ventricular tachycardia after exercise; recurrent ventricular tachycardia
episodes during dancing in a 12 year old girl; and a worsening intraventricular
conduction defect in a 15 year old girl, requiring pace-maker at the age of 22.
In the second group (n=3), a 16 year old asymptomatic boy presented with cardiac
arrest after running with no recurrence after implantable cardiac defibrillator;
a 14 year old boy with mild distal muscle weakness had atrial fibrillation; and
recurrent pre-syncope events due to paroxysmal atrial flutter were diagnosed in
a clinically unaffected 13 year old girl. A wide variation of CTG expansion was
observed (from 250 to 1200 repeats).
CONCLUSIONS: Severe cardiac symptoms may occur early in the course of DM1 in two
different manners. At various ages in congenital/childhood type affected
patients, as well as in clinically asymptomatic adolescents with minor muscle
signs. Moreover, cardiac manifestation may be the first isolated clinical sign
of DM1. No particular range of CTG expansion appear predictive. In addition to
our data, further prospective studies may be useful for accurate cardiac
management during infancy/adolescence as well as for genetic counselling in
young DM1 at risk individuals.
Supported By: Association Francaise contre les Myopathies (AFM)
[P03.130]
Clinical and Molecular Correlations in Myotonic Dystrophy Type 2
John W. Day, Kenneth Ricker, Katherine A. Dick, Laura Rasmussen, Joline C.
Dalton, Jennifer F. Jacobsen, Christina L. Liquori, Wolfram Kress, Christiane
Schneider, Melinda L. Moseley, Laura P. W. Ranum Minneapolis, MN; Wurzburg,
Germany
OBJECTIVE: To determine the breadth of clinical features in myotonic dystrophy
type 2, and to determine the correlation of those features with the size of the
tetranucleotide CCTG repeat expansion responsible for the disease.
BACKGROUND: Myotonic dystrophy (DM) causes a distinctive constellation of
seemingly unrelated clinical features that can be caused by mutations on either
chromosome 19 (DM1) or 3 (DM2). We recently demonstrated that a CCTG expansion
in intron 1 of ZNF9 causes DM2. We now report the clinical and molecular
correlations of this mutation in approximately 250 affected individuals from 82
families.
DESIGN/METHODS: Neurological and electrophysiological examinations of many
patients were carried out in the field, while others were studied in more detail
in clinic.
RESULTS: Individuals affected by DM2 have a complex clinical presentation that
is strikingly similar to DM1: a characteristic pattern of weakness involving
facial, neck flexor, finger flexor and hip girdle musculature; iridescent
cataracts that can be symptomatic in the 3rd decade of life; hypotestosteronism
and oligospermia in males; hypogammaglobulinemia, insulin insensitivity, and
elevated creatine kinase; potentially fatal cardiac involvement involving severe
arrhythmias or progressive cardiomyopathy. As in DM1, skeletal muscle biopsies
show the characteristic features of severe fiber atrophy and a profusion of
central nuclei, with intranuclear RNA inclusions in essentially all nuclei. We
investigated various aspects of disease severity and found no correlation with
CCTG repeat length. To date there are no known cases of severe congenital onset
of DM2, and the cognitive involvement appears to be much less severe than can
occur in DM1.
CONCLUSIONS: The DM1 CTG and DM2 CCTG expansions cause a strikingly similar
constellation of clinical features, electrophysiological abnormalities and
muscle histopathology including the presence of RNA foci. The clinical
similarities of DM1 and DM2 indicate that the downstream effects of
microsattelite expansions expressed in RNA cause the features common to both
disesases.
Supported By: NIH/NINDS NS35870-05
NIH/Univ MN GCRC
MDA
[P03.128]
The Prevalence of Myotonic Dystrophy in Israeli Jewish Communities:
Inter-Community Variation and Founder Premutations
Rival Segel, S. Silverstein, I. Lerer, E. Kahana, R. Meir, M. Sagi, N. Zilber,
Amos D. Korczyn, Y. Shapira, Z. Argov, R. Carmi, Zipora Falik, C. Ligum, Moshe
Frydman, Z. Gelman-Kahan, S. Shalev, Y. Finkelstein, Devora Abeliovich Jerusalem,
Israel; Israel; Tel-Aviv, Israel
OBJECTIVE: We attempted to perform a comprehensive epidemiological and genetic
survey of MyD among Jews living in Israel.
BACKGROUND: Myotonc dystrophy (MyD) occurs all over the world but exact data on
prevalence and frequency of premutations of the unstable DMPK-(CTG)n allele on
chromosone 19 in various populations are lacking.
DESIGN/METHODS: The frequency of unrelated sibships in each community was used
as an estimate of the transition rate from stable to unstable DMPK-(CTG)n
alleles assuming that each transition founds a new MyD sibship.
RESULTS: The average prevalence of myotonic dystrophy (MyD) was 15/105 with
inter-community variations; the Ashkenazi Jews had the lowest rate (5.7/105)
which was much higher among Yemenite Jews (47.3/105; p<0.05). This study
indicated that the differences in the prevalence of MyD are a result of higher
mutation rate in the non-Ashkenazi Jews (>50/108) as compared to the rate in
the Ahskenazi Jews (16.3/108). The intragenic haplotypes of the MyD alleles were
the same as in many populations over the world. However, two MyD closely linked
markers D19S207 and D19S112 were in linkage dysequilibrium with the MyD mutation
in patients of Yemenite and Moroccan extractions but not in Ashkenazi patients.
This observation indicated a common ancestral origin for the MyD premutation in
patients of a given ethnic origin.
CONCLUSIONS: The difference in the prevalence of MyD among Jewish communities is
a consequence of high frequency of founder premutations among non-Ashkenazi Jews,
and particularly Yemenite Jews.