MOLECULAR APPROACHES TO HUMAN NEUROMUSCULAR DISEASE 
Louis M. Kunkel, Ph.D., Associate Investigator 
The previous report detailed the complete clon- 
ing, sequencing, and identification of dystrophin, 
the protein product of the Duchenne/Becker mus- 
cular dystrophy (DMD/BMD) locus. This year has 
led to an increased understanding of dystrophin's 
function and how the absence of dystrophin in pa- 
tients with dystrophin deficiency might be cor- 
rected. Dystrophin had previously been shown to 
be expressed in all types of muscle and the nervous 
system. Recent work has shown that the protein is 
slightly different in these tissues and may function 
differently in those tissues that produce dystrophin. 
The transcription start point is different for the ner- 
vous system than for muscle. This second start 
point lies at least 100 kb away from the muscle start 
point and has complete activity in in vitro assays. 
There is currently a search for patients who might 
have disruptions of this new first exon. Among 
those patients with X-linked mental retardation but 
no muscle weakness, there may be some who have 
mutations of this first exon. 
One ongoing goal has been to relate molecular 
defects of the dystrophin gene with clinical symp- 
toms observed in patients. Over 400 patients with 
DMD and the milder BMD have been studied at the 
molecular level; nearly 95% of patients fit the ear- 
lier theory of frameshift or nonframeshift deletion 
mutations. As part of this study an additional 15 
exons from genomic DNA were sequenced, and oli- 
gonucleotides to detect more than 90% of all dele- 
tions by the rapid polymerase chain reaction have 
now been designed. Analysis of in-frame deletions 
found in BMD patients has established that the dif- 
ferent domains of the dystrophin protein are more 
or less sensitive to disruption. Deletion mutations 
in the central portion of the gene are predicted to 
result in very mild to no symptoms at all— very dif- 
ferent from classical DMD and BMD. Because of the 
association of cardiac problems with many of the 
DMD and BMD patients, a large collaborative effort 
has been established to screen males with cardiac 
problems for mutations in the central region, as 
well as other parts, of the dystrophin gene. 
Concordant with experiments to understand 
more of dystrophin's function has been the isola- 
tion of proteins that are related in structure to dys- 
trophin. These proteins may be capable of replac- 
ing dystrophin in DMD and BMD muscle and may 
interact with dystrophin in normal muscle. There is 
also another class of proteins that are part of the 
membrane cytoskeleton and might interact with or 
bind to dystrophin but are unrelated to dystrophin. 
Some of the relatives and binding proteins of dys- 
trophin have been identified, and these are cur- 
rently being isolated. It is hoped that this will lead 
to a better idea of the overall function of the mem- 
brane cytoskeleton in muscle and nerve. This line 
of experiments may have an additional benefit, in 
that these previously uncharacterized proteins are 
prime candidates to be disrupted in other neuro- 
muscular diseases. Both DNA samples and tissues 
from patients who exhibit clinical symptoms that 
differ from DMD and BMD but might represent 
disruptions of the genes encoding the dystrophin- 
related or dystrophin-binding proteins are cur- 
rently being banked by the laboratory. 
The last area of research in which the laboratory 
has had some promising results concerns treatment 
of neuromuscular diseases. An animal model of dys- 
trophin deficiency has been used, in collaboration 
with the laboratory of Dr. Terry Partridge in Lon- 
don, to transplant normal mouse muscle cells into 
the muscle of the dystrophin-deficient mouse. The 
input cells formed new muscle fibers and expressed 
dystrophin. The dystrophin was of normal size, lo- 
cation, and nearly 50% of normal levels. These 
promising experiments were done under special 
conditions; more experiments will be necessary to 
optimize the transplants so that they might be ap- 
plied to preliminary human experiments. The trans- 
plantation of muscle cells may have far-reaching 
consequences for therapy for other disorders. Any 
metabolite produced by muscle that is affected by a 
genetic disorder might be corrected by cell trans- 
plantation. 
In the next year. Dr. Kunkel's laboratory will at- 
tempt to address directly patient care and improved 
detection of the genetic disorders that affect both 
muscle and the nervous system. 
Dr. Kunkel is also Associate Professor of Pediatrics 
at Harvard Medical School. 
Continued 
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