Genetic Defects in the Metabolic Pathways 
Interconnecting the Urea 
and Tricarboxylic Acid Cycles 
David L. Valle, M.D. — Investigator 
Dr. Valle is also Professor of Pediatrics, Medicine, Molecular Biology and Genetics, and Biology at the 
Johns Hopkins University School of Medicine. He received both his undergraduate degree in zoology and 
his medical degree from Duke University. His internship and residency in pediatrics were completed at 
the Johns Hopkins Hospital. His postdoctoral research in metabolism was done at NIH. 
KJMAN biochemical genetics has been a 
ruitful area of study since its beginning 
with the work of Sir Archibald Garrod early in this 
century. Inherited defects in our body's chemis- 
try or, as Garrod called them, inborn errors of 
metabolism, are intrinsically interesting and 
serve as important models for all genetic diseases. 
My colleagues and I have been involved in the 
study of several aspects of these disorders, 
including clinical diagnosis, biochemical charac- 
terization, delineation of pathophysiologic mech- 
anisms, development of new therapeutic ap- 
proaches, and molecular studies of the involved 
genes. 
We have focused on disorders of amino acid 
metabolism, particularly those involving two 
fundamentally important areas of metabolism: 
the urea cycle, which is involved in the conver- 
sion of excess nitrogen from a toxic to a nontoxic, 
readily excreted form; and the tricarboxylic acid 
cycle, an essential component of energy metabo- 
lism. Recently we have extended these interests 
to include inborn errors in the biogenesis of the 
peroxisome, a ubiquitous, subcellular organelle 
that contains about 40 enzymes important in a 
variety of anabolic and catabolic processes. 
One of the amino acid disorders that we are 
studying intensively is an inborn error of orni- 
thine metabolism known as gyrate atrophy of the 
choroid and retina (GA). This progressive, blind- 
ing chorioretinal degeneration with associated 
cataract formation is inherited as an autosomal 
recessive trait. The primary biochemical defect is 
deficiency of the enzyme ornithine-6-aminotrans- 
ferase (OAT), which results in an approximate 
10-fold accumulation of ornithine in all bodily 
fluids. 
Despite the systemic nature of the metabolic 
abnormality in GA, the clinical phenotype is lim- 
ited to the eye. Thus GA is one of a very few iso- 
lated, inherited retinal degenerations for which a 
primary biochemical defect is known. In an ex- 
tensive molecular analysis of the OAT genes of 85 
probands from GA families around the world, we 
have detected 34 OAT mutations. Other investi- 
gators have added another 20, and together these 
54 OAT mutations account for 128 (75 percent) 
of the possible 170 mutant alleles in our patient 
population. 
This compilation of OAT mutations allows one 
to determine their consequences on the steady- 
state levels of OAT mRNA and on the structure 
and function of OAT protein in the patients' cul- 
tured skin fibroblasts or when expressed in a het- 
erologous system, Chinese hamster ovary cells, 
which lack endogenous OAT mRNA and protein. 
We find that more than 80 percent of the mutant 
alleles produce normal amounts of normally 
sized OAT mRNA. A small fraction (approxi- 
mately 10 percent) of mutant alleles, all with 
point mutations that truncate the open reading 
frame in the penultimate exon or earlier, have 
markedly reduced levels of OAT mRNA. In con- 
trast to their mRNA phenotype, approximately 80 
percent of the OAT mutant alleles, including at 
least 13 missense mutations, yield little or no de- 
tectable OAT antigen. Thus destabilization of the 
protein is the most common consequence of 
these mutations. However, two missense alleles, 
Rl 80T and Rl 54L, inactivate OAT function with- 
out reducing OAT antigen. We speculate that the 
involved residues may play a role in the active 
site of OAT. Studies are now in progress to pro- 
duce the large quantities of OAT necessary for 
x-ray crystallography to determine directly the 
consequences of these mutations in OAT struc- 
ture and function. A portion of this work on gy- 
rate atrophy is supported by a grant from the Na- 
tional Institutes of Health. 
The OAT-catalyzed reaction is an essential step 
in the metabolic pathway that interconnects the 
urea and tricarboxylic acid cycles and, as might 
be predicted, is subject to complex regulation. In 
liver, the regulation of OAT expression is coordi- 
nated with other urea cycle-related enzymes. We 
have identified a sequence motif in the 5'-flank- 
ing region of OAT that is also present in the pro- 
moters of several other urea cycle enzymes and 
have obtained evidence that this motif is a cis- 
acting element involved in the regulation of these 
genes. Surprisingly, localization of OAT expres- 
sion by in situ hybridization and immunohisto- 
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