HUMAN GENETICS AND MUTATIONAL MODELS 
C. Thomas Caskey, M.D., Investigator 
I. Lesch-Nyhan Syndrome. 
The complete DNA sequence of the hypoxan- 
thine guanine phosphoribosyltransferase (HPRT) 
gene has been determined by automated sequenc- 
ing and computer analysis. The method utilized 
"shotgun" sequencing, a method with the ad- 
vantage of sequence redundancy and accuracy, 
simplicity, and rapid data analysis. One continuous 
sequence of 58,000 base pairs (bp) was achieved 
by sequencing 250,000 bp. This sequence iden- 
tified a new class of minisatellite sequence with- 
in the HPRT gene with considerable polymor- 
phism. The sequence permitted development of 
a rapid polymerase chain reaction (PGR) multi- 
plex (many genetic sites) and simple nonradioac- 
tive method to identify mutations causative of 
Lesch-Nyhan (LN) syndrome. Automated DNA se- 
quencing of these mutations makes possible iden- 
tification of the new mutations in the germline 
causing LN in a specific family. These studies are 
now completed in 1-2 days rather than 7-14 days 
by earlier methods. Application to the prevention 
of disease by female carrier detection and prenatal 
diagnosis has been successfully achieved for 32 
families. 
II. Uricase. 
Humans are devoid of uricase activity and elimi- 
nate purines as uric acid, a cause of gout in some 
patients. The mouse possesses uricase activity and 
eliminates purines as allantoin. HPRT deficiency in 
humans leads to LN syndrome, while the defi- 
ciency in mice is without phenotypic effects. It is 
possible that uricase has a protective effect on 
HPRT-deficiency central nervous system damage. 
The uricase cDNA and gene of mice are character- 
ized. A nonfunctional gene in humans has been 
cloned. The functional cDNA of the baboon has 
been cloned and characterized. All have been com- 
pared. Two nonsense mutations were found in the 
human sequence that would render it nonfunc- 
tional. The highly conserved nature of the human, 
mouse, and baboon coding sequence suggest the 
mutation in humans is very recent. Attempts to cre- 
ate the uricase'/HPRT" mouse are now proceeding, 
using the embryonic stem cells of the mouse and 
insertional mutagenesis via homologous recombi- 
nation. 
III. Duchenne Muscular Dystrophy. 
Duchenne muscular dystrophy (DMD) is a severe 
X-linked muscular dystrophy that occurs by new 
mutations, which are largely (85%) deletions and 
duplications of portions of the 2.5 million bp gene. 
A simple nonradioactive scanning DNA detection 
method has been developed and validated by an in- 
ternational multicenter collaboration. The PGR 
multiplex method uses DNA sequence information 
on the DMD gene mutation "hot spots" (prone to 
deletion and duplication) and presently includes 
nine positions. This collaborative study found the 
method capable of diagnosing 80% of all gene du- 
plications and deletions. Fluorescent detection 
methods have made quantitation and application to 
detection of female carriers possible. This simple di- 
agnostic approach for DMD is now the standard of 
practice for diagnosis. 
The identification of the mutations in the DMD 
gene of mice that cause mdx phenotype is under 
way. This is made possible by the complete cloning 
and sequencing (75%) of the mouse dystrophin 
cDNA. The mdx mutation in the Bulfield mutant is 
a nonsense mutation at a position early in the gene. 
The two mdx mutants developed in collaboration 
with Dr. Verne Ghapman by germline mutation are 
under study for their mutation. 
lY Ornithine Transcarbamylase Deficiency. 
Ornithine transcarbamylase (OTG) deficiency is 
the most common urea cycle disorder in humans 
and leads to severe neonatal coma and death. A 
new rapid scanning method, which is based on 
knowledge of the OTG DNA sequence, has been de- 
veloped for the mutations. Mutations are detected 
by cleavage at the site following heteroduplex for- 
mation (wild-type sequence :mutant sequence) and 
chemical cleavage. The approach has the advantage 
of speed, ease, and scanning over large DNA seg- 
ments. The unique mutations of seven families 
were identified, and this knowledge was used to di- 
agnose female carriers and conduct prenatal diag- 
nosis. 
Genetic correction of the OTG-deficient sparse 
fur (spf) mouse has been achieved by transgenic 
approaches. A recombinant transgene consisting of 
the human OTG cDNA under the control of an 800 
bp mouse promoter was used; single-cell embryos 
Continued 
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