MOLECULAR GENETICS OF HUMAN DISORDERS 
Savio L. C. Woo, Ph.D., Investigator 
Dr. Woo's laboratory is interested in phenylketo- 
nuria (PKU), a genetic disorder that predisposes 
affected children to develop severe mental retarda- 
tion. The disorder is secondary to a genetic defi- 
ciency of hepatic phenylalanine hydroxylase (PAH). 
It is transmitted as an autosomal recessive trait and 
has a prevalence of ~1 in 10,000 Caucasians. 
I. Molecular Basis and Population Dynamics of PKU. 
A. Molecular lesions of prevalent mutant PAH al- 
leles in Caucasians. The PAH gene represents a 
highly polymorphic locus in the human genome, 
with more than 50 established restriction fragment 
length polymorphism (RFLP) haplotypes. In PKU 
patients throughout Europe, 75% of mutant PAH al- 
leles are associated with haplotypes 1-4. The mo- 
lecular lesions associated with mutant haplotypes 2 
and 3 were characterized previously and shown to 
be in linkage disequilibrium with their respective 
haplotypes throughout the European continent. 
The results suggested a "founder effect" for the 
spread of PKU among Caucasians. More recently, 
the PAH locus of a compound heterozygote bearing 
mutant haplotype 1 and 4 alleles was analyzed by 
polymerase chain reaction (PCR) amplification of 
individual exons, followed by sequencing analysis. 
Two missense mutations were observed, and both 
were Arg to Gin substitutions. The mutation involv- 
ing residue 158 is associated with mutant haplo- 
type 4, while that in residue 261 is associated with 
haplotype 1. The linkage disequilibrium is again 
maintained throughout the European continent, 
supporting the concept of founder effect as the 
cause of spread of PKU in Caucasians. In this case, 
however, not all mutant haplotype 1 and 4 alleles 
are represented by these two mutations. The obser- 
vation is not surprising, because haplotypes 1 and 4 
are prevalent among normal alleles and could sus- 
tain multiple mutations during human evolution. 
B. Recurrent mutations in the PAH locus. A Glu^*^ 
to Lys^^° mutation has previously been observed in 
PKU patients from northern Africa; this mutation is 
in linkage disequilibrium with haplotype 38. The 
same mutation has recently been observed in two 
different families from northern Europe. One such 
mutant allele is associated with haplotype 1; the 
other is associated with haplotype 4. Because the 
three haplotypes are different in their individual 
RFLP distributions, the results suggest that recur- 
rent mutations at the same site of the PAH gene 
might have occurred on different background chro- 
mosomes in different regions of the world. Exami- 
nation of the nature of the mutation revealed a 
transversion event involving a CpG dimer, which is 
known to be a methylation site and more suscepti- 
ble to mutations. 
C. Molecular genetics of PKU in Orientals. Al- 
though PKU was originally thought to be a disorder 
restricted to Caucasians, recent implementation of 
a PKU-screening program in China showed that the 
disorder is also prevalent among Orientals (1 in 
16,000). In that population, however, the majority 
of normal as well as mutant PAH alleles are associ- 
ated with a single haplotype, haplotype 4. Conse- 
quently, RFLP haplotyping in that population is not 
as informative as it is in Caucasians. Molecular anal- 
ysis of the mutant alleles by PCR amplification and 
direct DNA sequencing has revealed three distinct 
missense mutations: Arg^^^ to Ter^^\ Tyr^^"* to 
Cys^'^'*, and Arg'*^^ to Pro^^^. These three mutations 
are in linkage disequilibrium with mutant haplo- 
type 4 and together represent ~30% of all PKU 
chromosomes in the Oriental population. The re- 
sults provide conclusive evidence that multiple mu- 
tations occurred on the same haplotype back- 
ground in the PAH locus and that there are 
prevalent mutant alleles in the Oriental popula- 
tion. Thus carrier detection may be possible in that 
population as well. 
D. PKU mutations occurred after racial diver- 
gence. Oligonucleotides corresponding to the prev- 
alent mutant alleles in the Caucasian and Oriental 
populations were used to analyze PKU patients 
from both continents. These mutant alleles are 
non-overlapping, providing conclusive evidence 
that the majority of PKU mutations must have oc- 
curred on the PAH locus in humans after racial di- 
vergence. 
II. Somatic Gene Therapy of PKU. 
The construction of a recombinant retrovirus 
bearing the human PAH cDNA under the transcrip- 
tion regulation of a liver-specific promoter has been 
reported previously. The recombinant retrovirus 
was capable of infecting primary mouse hepato- 
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