MOLECULAR APPROACHES TO HUMAN NEUROMUSCULAR DISEASE 
Louis M. Kunkel, Ph.D., Investigator 
Previous reports have detailed the identification 
of dystrophin as the protein altered by mutation to 
yield Duchenne and Becker muscular dystrophy. In 
an effort to characterize the consequences of abnor- 
mal dystrophin in diseased tissues, Dr. Kunkel and 
his colleagues have refined their analysis of the dis- 
tribution of dystrophin in those tissues that nor- 
mally express it and have continued to characterize 
some of the nondeletion mutations that yield abnor- 
mal dystrophin. To further the understanding of dys- 
trophin function and its disruption in disease, the 
laboratory has made a major effort to characterize 
additional cytoskeletal proteins that might act in 
tandem with dystrophin in muscle tissue. These 
dystrophin-related proteins might play some role in 
mitigating the consequences of absent dystrophin 
and are themselves prime candidates to be altered in 
other neuromuscular diseases. 
The 427-kDa form of dystrophin has been shown 
in this and other laboratories to be expressed in 
smooth, cardiac, and skeletal muscle, as well as in 
the brain of both humans and mice. In collaboration 
with Dr. Simon Watkins, Dr. Kunkel's group has re- 
fined this localization with a sensitive and specific 
antibody directed against a large fusion peptide en- 
compassing the last half of dystrophin. With this an- 
tibody the laboratory has shown, contrary to pre- 
vious biochemical fractionations, that dystrophin in 
skeletal muscle is not associated with any internal 
cellular structures, but rather is solely localized to 
the plasma membrane. 
An elevated concentration of dystrophin is found 
at the myotendinous junction and the neuromuscu- 
lar junction, the labeling in the latter being more 
intense in the troughs of the synaptic folds. As in 
skeletal muscle, dystrophin is concentrated at the 
surface plasma membrane of cardiac muscle but is 
notably absent from the membrane areas that overlie 
the adherence junctions of the intercalated discs. 
Much less plasma membrane labeling is observed in 
smooth muscle and is concentrated in areas of the 
membrane underlain by membranous vesicles. A 
previous report described the postsynaptic mem- 
brane labeling of Purkinje cells of the cerebellum 
and a subset of cortical neurons of the cerebral 
cortex. 
Dr. Kunkel's group has continued the character- 
ization of alternative dystrophin mutations, espe- 
cially those involved in splicing of dystrophin's 
huge primary transcript. The aberrant splicing of at 
least one exon has been documented, but the exact 
mutation remains elusive. Despite extensive se- 
quences surrounding the exons involved, no nu- 
cleotide change has been documented as the cause 
of abnormal splicing. 
The most exciting findings related to alternative 
exon usage have probably been the demonstration 
by this and other laboratories of two alternative tran- 
scription start points near the end of the gene that 
yield shortened dystrophin proteins lacking the 
amino-terminal domain and much of the rod domain 
of the protein. One of these begins transcription in 
the intron before exon 62 (work of Dr. David Yaff'ee 
in Israel) and encodes a 71-kDa protein. Dr. Kun- 
kel's group has detected this protein with carboxyl- 
terminal antibodies and, like the Israeli group, finds 
it in many tissues, but not in muscle. Dr. Kunkel and 
his colleagues have also detected all 5-kDa protein 
with their carboxyl-terminal antibodies that was 
originally thought to be a related protein but has 
proved to be encoded from a transcription start 
point between exons 53 and 54 of the dystrophin 
gene. This protein is produced only by Schwann 
cells and has been detected in no other tissues. In- 
vestigation of the function of these two shorter dys- 
trophin proteins and their pathogenic role, if any, 
continues. 
One of the major efforts of the laboratory over the 
past year has been to identify the gene that is mu- 
tated to yield the degenerative motor neuron disease 
spinal muscular atrophy (SMA). An antigenically 
cross-reactive protein was detected in human and 
mouse brain with one of the laboratory's carboxyl- 
terminal dystrophin antibodies. The human form of 
this protein was cloned from an expression cDNA 
library, and when the human sequence was used to 
screen the Gene Bank database, the encoded protein 
was found to be that of the previously cloned micro- 
tubule-associated protein IB (MAP- IB). The human 
locus for this protein was found to be chromosome 
5 , the location of mutations causing SMA. 
The laboratory identified two new dinucleotide 
repeat polymorphisms in the human MAP- IB locus, 
and these were shown to be linked very tightly to 
SMA mutations in SMA families. The tight linkage of 
MAP- IB to SMA, together with the known function 
of mouse MAP- IB in neuronal survival and out- 
growth, made this a possible candidate for the SMA 
gene. The entire 1 0-kb human transcript was cloned 
as cDNA, and the complete sequence of the human 
gene determined. No gross structural alterations 
were found in the MAP- IB gene in 40 unrelated SMA 
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