MOLECULAR GENETICS AND GENE THERAPY FOR METABOLIC DISORDERS 
Savio L. C. Woo, Ph.D., Investigator 
Dr. Woo and his colleagues are interested in study- 
ing phenylketonuria (PKU) , a genetic disorder that 
predisposes affected children to develop severe 
mental retardation. The disorder is secondary to a 
genetic deficiency of the enzyme hepatic phenylala- 
nine hydroxylase (PAH) . It is transmitted as an auto- 
somal recessive trait and has a prevalence of ~ 1 in 
10,000 Caucasians and 1 in 16,500 Orientals. 
Molecular Basis and Population 
Dynamics of PKU 
The establishment of multiple RFLP (restriction 
fragment length polymorphism) sites in the human 
PAH (hPAH) gene and its use in prenatal diagnosis of 
PKU have been reported previously. The 3-allelic 
Hindlll RFLP system in the hPAH gene was shown to 
be caused by an AT-rich (70%) minisatellite region. 
This region contains various multiples of a 30-bp 
tandem repeat and is located 3 kb downstream of the 
final exon of the gene. Characterization of this vari- 
able-number tandem repeat (VNTR) region by PGR 
(polymerase chain reaction) amplification indi- 
cated that the previously reported 4.0-kb Hindlll 
allele contains 3 such repeat units, and the 4.4-kb 
Hindlll allele contains 12 repeats. On the other 
hand, the 4.2-kb Hindlll fragment can contain 6-9 
copies of this repeat, which permit more detailed 
analysis of mutant chromosomes bearing a 4.2-kb 
allele. 
Kindred analysis in PKU families demonstrated 
Mendelian segregation of the VNTR alleles, as well 
as associations between these alleles and certain ma- 
jor PKU mutations. For example, the R261Q muta- 
tion is associated almost exclusively with an allele 
containing 8 repeats, and the IVS-10 mutation with 
one containing 7 repeats. The combined use of this 
VNTR system and the existing RFLP haplotypes in- 
creases substantially the performance of prenatal 
diagnostic tests for PKU. In addition, this VNTR will 
be most useful in studies concerning the origins and 
distributions of prevalent PKU mutations in various 
human populations. 
Tissue-Specific Expression 
of the hPAH Gene 
In primates PAH is expressed specifically in the 
liver. In rodents PAH activity is also present in the 
kidney, although at a much lower level. A 9-kb geno- 
mic DNA fragment flanking the 5' end of the hPAH 
gene was fused to the bacterial gene for chloram- 
phenicol acetyltransferase (CAT). The hPAH-CAT 
minigene was used to generate multiple transgenic 
mouse lines. In all expressing lines, CAT activity 
was detected predominantly in the liver and, at 
much lower levels, in the kidney. 
By immunohistochemical staining, CAT expres- 
sion was localized to hepatocytes and renal epithe- 
lial cells, both of which also express the endoge- 
nous mouse PAH gene. Furthermore, both the 
transgene and the endogenous mouse PAH gene 
were activated at about the same stage of embryonic 
development in the mouse liver. These results sug- 
gest that the 9-kb DNA fragment flanking the 5' end 
of the hPAH gene contains all necessary cis-acting 
elements to direct its tissue-specific expression and 
development regulation in vivo. 
The 3 1 9-bp region immediately flanking the initi- 
ation site of the gene is characterized by the lack of a 
proximal TATA box and the presence of sequences 
homologous to GC boxes, CACCC boxes, CCAAT 
boxes, activator protein 2-binding sites, glucocorti- 
coid, and cAMP response elements. When this short 
DNA fragment was used to direct the transcription of 
a reporter gene, specific expression in hepatoma 
cells was observed. The results suggest that the 
hPAH gene has a TATA- less promoter regulated by 
multiple transcription factors and that the system 
will permit the identification and characterization 
of mutations in the promoter region of the hPAH 
gene that cause PKU. (This portion of the work was 
supported in part by a grant from the National Insti- 
tute of Child Health and Human Development, Na- 
tional Institutes of Health.) 
Somatic Gene Therapy 
of Hepatic Deficiencies 
The construction of a recombinant retrovirus 
bearing hPAH cDNA under the transcription regula- 
tion of a liver-specific promoter was reported previ- 
ously. The recombinant retrovirus was capable of 
transducing primary mouse hepatocytes in culture, 
and hPAH mRNA accumulated in the cells to a level 
comparable to that present in normal human liver. 
Dr. William Dove's laboratory at the University of 
Wisconsin recently created a mouse strain deficient 
in PAH that has provided an excellent model system 
for exploring the possibility of its phenotypic cor- 
rection by hepatic gene therapy. A recombinant ret- 
rovirus containing mouse PAH cDNA was con- 
structed and used to transduce hepatocytes isolated 
from the PAH-deficient mice. 
GENETICS 289 
