MEDICAL GENETICS AND MODELS OF MUTATION AND THERAPY 
C. Thomas Caskey, M.D., Investigator 
Discovery of Fragile X Syndrome 
and Myotonic Dystrophy Mutations 
Fragile X syndrome is an X-linked recessive men- 
tal retardation disorder. The discovery of the muta- 
tion responsible for fragile X syndrome, an expand- 
ing CCG triplet repeat, gave insight into the 
molecular basis of inherited diseases with the fea- 
ture of ' ' anticipation . ' ' Expansion of the CCG repeat 
occurred from generation to generation within fami- 
lies once the repeat number reached approximately 
52. This triplet expansion led to decreased expres- 
sion of the FMR- 1 {fragile X mental retardation 1) 
gene once the repeat number exceeded approxi- 
mately 200. Recently, both FMR-1 mRNA and pro- 
tein were found to be absent in fragile X syndrome. 
These discoveries collectively document a new mu- 
tational mechanism for humans (repeat sequence 
expansion) and identified the gene associated with 
the most common form of human mental retarda- 
tion, fragile X syndrome. 
Myotonic dystrophy, the most common severe my- 
opathy of adults, is inherited in an autosomal domi- 
nant manner. "Anticipation" is a well-documented 
feature of myotonic dystrophy. Severe and fre- 
quently lethal infantile myotonia occurs in children 
of mothers (not fathers) with mild myotonic dys- 
trophy. The myotonic gene was mapped to an ~ 1 - 
Mb region of chromosome 1 9 by a consortium spon- 
sored by the Muscular Dystrophy Association. 
Knowledge of the molecular basis of anticipation in 
fragile X syndrome made possible development of a 
simple scanning method for identification of an un- 
stable triplet repeat within the myotonic dystrophy 
locus. This method identified both the myotonic 
dystrophy mutation (an AGC repeat) and the gene, 
myotonin protein kinase. Using simple diagnostic 
methods capable of detecting triplet expansion, fam- 
ilies and physicians now have accurate DNA-based 
diagnostics for myotonic dystrophy. Recombinant 
bacterial methods allowed a simple means of synthe- 
sis and purification of myotonin protein kinase. 
DNA sequencing of skeletal and muscle forms indi- 
cates that different forms of the gene are expressed. 
Because protein kinases are involved in the regula- 
tion of target proteins, research efforts are now di- 
rected toward elucidation of the pathogenesis of 
myotonic dystrophy, including study of target pro- 
tein (s) and effect on muscle function. 
The scanning method developed for triplet re- 
peats in the myotonic dystrophy gene is being used 
to initiate a search for other disease genes. Approxi- 
mately 50 independent genes have been isolated 
that contain triplet repeats similar to those found in 
fragile X syndrome, myotonic dystrophy, and spino- 
bulbar muscular atrophy. Such triplet sequences are 
highly mutable; these genes may therefore be asso- 
ciated with additional heritable diseases. The scan- 
ning methods in progress may rapidly identify these 
diseases. 
Duchenne Muscular Dystrophy: 
Cause and Treatment 
Rapid diagnostic methods for Duchenne muscular 
dystrophy (DMD) indicate that 75% of all mutations 
involve deletions, the endpoints of which are fre- 
quently found in intron 44 of the dystrophin gene. 
Approximately 38,000 base pairs of this mutational 
hotspot have been determined. A rapid polymerase 
chain reaction (PCR) method can be used to define 
the deletion site in individual patients to within 
5,000 bp of this 2,400,000-bp gene. The sequence 
of the region has revealed three sequence repeats of 
unusual character, as well as a highly polymorphic 
simple repeat sequence. These regions are suspect 
for the hotspot deletion sites. Computer analysis 
and subsequent mRNA studies have identified an ad- 
ditional and unexpected gene within intron 44, ori- 
ented in the opposite direction to dystrophin. 
Efforts to correct DMD defects in patients are di- 
rected toward gene transfer therapy using a mini- 
gene constructed from full-length mouse dystro- 
phin cDNA. This minigene has succeeded in the 
correction of the mdx mouse deficiency of dystro- 
phin when regulated by a muscle-specific promoter 
and injected into the single-cell mouse embryo. 
Such a correction proves the utility of the minigene, 
but this strategy is not useful in humans. Viral deliv- 
ery methods are under study for correction of the 
human disease. Retroviral and adenoviral vectors 
cannot accommodate the 1 1,000-bp minigene, and 
thus truncated versions of the full-length cDNA are 
being constructed. A 3,000-bp section of the spec- 
trin repeat was removed in one construction; a 
5,000-bp region was removed in a second. Fur- 
thermore, 2,500 bp were removed from the 3'- 
untranslated region of both minigenes. Each of the 
four truncated minigenes is being inserted into both 
adenoviral and retroviral vectors for study of dystro- 
phin expression. In preparation for human studies, 
primary muscle cells have been isolated from eight 
DMD patients. This project was supported by a grant 
from the Muscular Dystrophy Association. 
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