Human Disease Gene Identification 
and Correction 
C. Thomas Caskey, M.D. — Investigator 
Dr. Caskey is also Professor of Molecular Genetics, Biochemistry, Medicine, and Cell Biology at Baylor 
College of Medicine. He received his M.D. degree at Duke University. His internship and residency training 
were in internal medicine, also at Duke; his postdoctoral training was at NIH, under the supervision 
of Marshall Nirenberg. Dr. Caskey is a past president of the American Society for Human Genetics 
and was named Distinguished Service Professor by the Board of Trustees of Baylor. 
MOLECULAR genetics offers unprecedented 
opportunities for the discovery of disease 
genes, the development of simple DNA-based 
diagnostics, and correction of single-gene de- 
fects. Recent isolation of the gene responsible for 
the fragile X syndrome has led to improved diag- 
nostic procedures and discovery of a novel mech- 
anism for the occurrence of genetic disease. Fur- 
ther knowledge regarding the genetic mechanism 
and pathology of two other X chromosome dis- 
orders has also been gained. In addition, our labo- 
ratory has made significant progress in the devel- 
opment of gene replacement therapies for three 
diseases. 
Disease Gene Identification and Diagnosis 
The gene that contains the breakage point in 
fragile X syndrome has been isolated as the result 
of a collaboration with Stephen Warren (HHMI, 
Emory University) and Ben Oostra (Erasmus Uni- 
versity). This gene contains a three-nucleotide 
sequence repeated in tandem, and the repeat re- 
gion forms the fragile site itself. The number of 
repeats is variable and correlates with occurrence 
of the fragile X disorder. Normal individuals have 
approximately 6-50 repeats; affected individ- 
uals, over 200 repeats; and those with an inter- 
mediate number appear to harbor a "premuta- 
tion" that is very likely to give rise to the full 
mutation by inheritance through females. This 
phenomenon of genetic disease caused by se- 
quence amplification was previously unknown, 
but may prove to play a part in other inherited 
disorders. 
Since the length of this particular region of 
DNA is an indicator of the affected or carrier sta- 
tus of the individual, DNA diagnosis of fragile X 
syndrome can be performed by Southern analysis, 
and more recently by the polymerase chain reac- 
tion. These DNA-based methods improve on cyto- 
genetic diagnosis, particularly in the detection of 
female carriers and unaffected males who never- 
theless can transmit the disorder. 
Study of Genetic Disorders 
The central region of the dystrophin gene is a 
"hot spot" for the deletion end-points causing 
Duchenne and Becker muscular dystrophies. This 
region was studied in greater detail to determine 
possible mechanisms that could explain why de- 
letions were so frequent. In two independent pa- 
tients with Duchenne muscular dystrophy, the 
ends of deletion lay within a transposable ele- 
ment (a portion of DNA that can move around the 
genome) in the dystrophin gene. It is likely that 
such transposable elements are involved in other 
deletions in this and other genetic disorders. 
Lesch-Nyhan syndrome in humans results from 
lack of the enzyme hypoxanthine guanine phos- 
phoribosyltransferase (HPRT). The neurological 
aspects of this incurable disorder are poorly un- 
derstood. Study of the syndrome and develop- 
ment of therapeutic measures would greatly ben- 
efit from the generation of an animal model. 
Transgenic mice have been engineered to lack 
the uricase enzyme (which is absent in humans), 
and these are being bred with mice lacking the 
HPRT enzyme. This strategy has potential for gen- 
erating a mouse model of the human Lesch-Nyhan 
syndrome. 
Genetic Correction of Inlierited Disease 
Human genes can now be cloned, placed into 
defective (safe) viral vectors, and transferred into 
other cultured cells, embryonic cells, and ani- 
mals. These encouraging developments increase 
the likelihood of successful gene replacement 
therapy. Our laboratory is developing technology 
toward that objective for three heritable diseases. 
Each disease offers different technical and strate- 
gic challenges. 
Adenosine deaminase (ADA) deficiency is an 
inherited autosomal recessive disease. Bone 
marrow transplantation is curative, but carries 
the risk of a graft rejection . Administration of PEG 
ADA (the enzyme ADA attached to polyethylene 
glycol) on a continuing basis has provided im- 
provement in the immunologic function of pa- 
tients, but is not a cure. 
The goal of this laboratory is the development 
of a clinical gene therapy protocol for ADA defi- 
ciency. This will require the treatment of human 
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