Molecular Genetics of Neuromuscular Disease 
Louis M. Kunkel, Ph.D. — 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. 
WE continue to study the underlying cause 
of Duchenne and Becker muscular dystro- 
phies. Our identification of the gene led rapidly 
to our description of the encoded protein dystro- 
phin, which was found to be an unknown 
member of a family of proteins that includes the 
spectrins and a-actinins. Located at the inner face 
of the plasma membrane of myofibers, dystro- 
phin is thought to confer strength to the mem- 
brane during muscle contraction and relaxation. 
Absence or abnormality of dystrophin at this loca- 
tion causes the myofiber degeneration of Du- 
chenne and Becker dystrophy. Our work has led 
to improved diagnosis of the diseases and to test- 
able ideas on therapeutic intervention. 
Over the past year the laboratory has concen- 
trated on identifying new members of the dystro- 
phin family of proteins, in the hope that these can 
play a role in mitigating disease caused by dystro- 
phin deficiency. These dystrophin-related pro- 
teins might themselves be involved in other 
neuromuscular diseases. By antigenic cross- 
reactivity, we have cloned the human microtu- 
bule-associated protein IB (MAP- IB) and have 
mapped this locus in close proximity to muta- 
tions on chromosome 5 that cause a motor neu- 
ron degenerative disease, spinal muscular atro- 
phy (SMA). 
We have sequenced this human gene and have 
begun its direct mutational analysis in SMA pa- 
tients. We have also identified at the locus two 
separate (CA)n repeat polymorphisms that have 
been shown, in collaboration with Conrad 
Gilliam, to be linked tightly to SMA mutations. In 
this genetic analysis, a few rare recombinations 
have been detected between SMA and MAP- IB, 
implying that MAP- IB may be very close, but not 
involved in the disease. As a result of this observa- 
tion, we have cloned large segments of human 
DNA from the region of chromosome 5 as yeast 
artificial chromosomes (YACs) and have begun a 
search for other genes near MAP- IB, any of which 
might be the SMA gene. 
By reduced stringency hybridization with dys- 
trophin cDNA clones, a chromosome 6-encoded 
protein was identified by Kay Davies in Oxford. 
This dystrophin-related protein (DRP) is highly 
similar to dystrophin, and antibodies against it 
developed in our laboratory have revealed that 
the two proteins are almost identical in size. Im- 
munolocalization of DRP has shown that it colo- 
calizes with dystrophin in a developing myofiber. 
In a mature fiber, however, it is located only at 
the neuromuscular and myotendinous junctions 
of muscle. We are currently attempting to clone 
the entire coding sequence of DRP as cDNA. We 
believe that the sequence of this protein might 
reveal why it has such a specialized location in 
mature muscle and what role, if any, it might play 
in mitigating some of the effects of dystrophin 
absence in Duchenne dystrophy. We are also 
searching for other neuromuscular diseases that 
might be caused by DRP abnormalities. 
One obvious set of dystrophin relatives for us 
to attempt to characterize were the a-actinins. A 
smooth muscle form had been cloned and local- 
ized to chromosome 14, but there was at least 
one other that had been identified from chicken 
muscle. Using a conserved motif of the known 
a-actinin amino acid sequence, we designed a de- 
generate set of PGR (polymerase chain reaction) 
primers that amplified a human muscle product 
with the sequence of an a-actinin. Using this 
small product as a hybridization probe, we 
screened a muscle cDNA library and obtained two 
classes of hybridizing human cDNA clones. Se- 
quence analysis revealed that the clones con- 
tained two different a-actinins that were unique 
for humans. They are both muscle specific and 
are encoded from chromosomes 1 and 1 1 , respec- 
tively. We are now developing antisera specific 
for each of the muscle a-actinin gene products 
and attempting to identify disease phenotypes 
that might involve a-actinin. 
Abnormalities of dystrophin are easily detected 
at the protein level, and nearly 70 percent of the 
mutations that cause them have been shown to be 
deletions or duplications of some part of this ex- 
tremely large locus. We have designed primers 
from dystrophin's nucleotide sequence to allow 
PGR amplification of specific regions of dystro- 
phin's transcript. In analyzing these PGR prod- 
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