Molecular Genetics of Neuromuscular Disease 
Louis M. Kunkel, Ph.D. — Associate Investigator 
Dr. Kunkel is also Professor of Pediatrics and of Genetics at Harvard Medical School. He received his B.A. 
degree from Gettysburg College and his Ph.D. degree in biology from the Johns Hopkins University. He 
took postdoctoral training with Brian McCarthy at the University of California, San Francisco, and with 
Samuel Latt at the Children's Hospital, Boston. He held appointments at Children's Hospital/Harvard 
Medical School before joining HHMI. His honors include the Gairdner Award and election to the National 
Academy of Sciences. 
OVER the years our laboratory has worked on 
how abnormalities of the protein dystrophin 
result in the clinical symptoms of Duchenne and 
Becker muscular dystrophy. From our initial 
identification of the dystrophin gene, we have 
turned to the normal protein function and to how 
this might be restored to mitigate disease symp- 
toms. Our efforts have centered on proteins re- 
lated to dystrophin by structure or function. Not 
only may these replace absent dystrophin in dis- 
eased muscle, but they are themselves prime can- 
didates for disease-producing alteration. 
Over the past year we cloned two dystrophin 
relatives and studied their function in diseased 
and normal persons. We also designed more accu- 
rate ways of detecting mutations within the dys- 
trophin gene and pinpointed dystrophin in the 
central nervous system. We have also hypothe- 
sized a role abnormal dystrophin might play in 
cognitive function. 
After dystrophin had been cloned and se- 
quenced, it was seen to belong to a family of pro- 
teins with cytoskeletal function. A group at Ox- 
ford University, using clones for dystrophin, was 
able to isolate a part of a member of the family. 
This protein, encoded by a locus on chromosome 
6, was partly sequenced and shown to be 85 per- 
cent identical to dystrophin. 
Using the published sequence, we cloned a 
part of this locus and expressed the protein in 
bacterial cells. Antibodies directed against the 
new protein were produced. Used as a probe 
against normal muscle, the antibodies detected a 
protein the same size as dystrophin, and it was 
also found in the muscle of Duchenne patients. 
We called it dystrophin-related protein, or DRP. 
Using the antibodies to probe for DRP, we were 
able to show that many different tissues ex- 
pressed it and that the highest levels were in de- 
veloping fetal muscle. By microscopic localiza- 
tion, we showed further that DRP colocalized in 
muscle with the acetylcholine receptor. In a de- 
veloping muscle the protein colocalizes with 
dystrophin, but as the muscle matures, DRP mi- 
grates along with the acetylcholine receptor to 
the neuromuscular junction. 
We are currently trying to unravel why a pro- 
tein that is so similar to dystrophin has such a 
specialized function in muscle and how we might 
interfere with normal function to make DRP work 
more like dystrophin. We are also searching for a 
neuromuscular disease that might be caused by 
abnormalities of DRP. 
Our antibodies directed against dystrophin de- 
tected a second protein, which we have identi- 
fied and partly cloned. This large molecule was 
found exclusively in the brain. Using our dystro- 
phin antibodies as a probe, we were able to clone 
pieces of cDNA that encoded this protein. As part 
of our strategy in characterizing new members of 
the dystrophin family, we mapped the encoding 
locus to chromosome 5. A search of the human 
linkage map revealed that the gene altered to 
cause spinal muscular atrophy has been localized 
to chromosome 5ql 2. We have initiated a collab- 
orative effort with David Ward (Yale University) 
to localize further our dystrophin-related protein 
on chromosome 5 . We are also attempting to gen- 
erate highly polymorphic markers surrounding 
the locus to map this gene relative to SMA muta- 
tions. This latter effort will be in collaboration 
with Conrad Gilliam (Columbia University) , who 
was involved in the original mapping of SMA to 
chromosome 5. 
One of the curious features of some Duchenne 
patients is mental impairment. Because virtually 
all patients have almost no dystrophin, it was dif- 
ficult to explain the impairment in some. We 
showed that dystrophin was expressed in the 
brain, but under different regulatory control 
from that found in muscle. In the course of devel- 
oping more efficient dystrophin-detecting anti- 
bodies, we found one that was highly sensitive for 
dystrophin and showed little cross-reaction with 
other brain proteins. 
We used this antibody to localize dystrophin in 
the brain, both with the light microscope and the 
electron microscope. Unlike results in muscle, 
where all types of muscle reveal dystrophin, it 
was only found in a subset of neurons of the cere- 
bellum and cerebral cortex. We hypothesize that 
an absence of dystrophin in the cortical neurons 
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