Hg. Eiken et al., PKU MUTATION G46S IS ASSOCIATED WITH INCREASED AGGREGATION AND DEGRADATION OF THE PHENYLALANINE-HYDROXYLASE ENZYME, Human mutation, 7(3), 1996, pp. 228-238
The G46S mutation in the phenylalanine hydroxylase (PAH) gene was iden
tified by fluorescence-based single strand conformation polymorphism (
F-SSCP) analysis on phenylketonuria (PKU) haplotype 5,9 alleles. DNA s
equencing of PAH exon 2 revealed a G-to-A transition in cDNA position
136. G46S mutations were present on 17 of 236 Norwegian PKU alleles (7
.2%) and on 8 of 176 Swedish PKU alleles (4.5%). Analysis of all 13 ex
ons with the flanking regions further detected a 1316-35c>t polymorphi
sm (PAH intron 12), associated with both G46S and haplotype 5.9. Three
patients were homozygous for the G46S mutation, two were untreated an
d had mild and severe mental retardation, respectively. The G46S mutat
ion was introduced in the PAH cDNA by site-directed mutagenesis and ex
pressed in three different systems (the pMAL/Escherichia coli system,
the pcDNAS/human embryonic kidney (A293) cells, and the pcDNA3/TnT cou
pled in vitro transcription-translation system). The mutant recombinan
t E. coli fusion protein was recovered in high yield and with a specif
ic activity of the purified tetrameric form, which was higher than the
wild-type activity. After transient expression in A293 cells, the amo
unt of the G46S protein was only about 3% of the wild type at equal PA
H mRNA levels. The fusion protein cleaved by restriction protease fact
or Xa, as well as the enzyme produced by in vitro transcription-transl
ation, revealed an abnormal sus susceptibility to form catalytically i
nactive high molecular mass aggregates of the enzyme, This aggregation
, followed by an increased cellular degradation of the G46S mutant enz
yme, is compatible with the clinical/metabolic phenotype of the affect
ed homozygous and compound heterozygous patients. (C) 1996 Wiley-Liss,
Inc.