PKU MUTATION G46S IS ASSOCIATED WITH INCREASED AGGREGATION AND DEGRADATION OF THE PHENYLALANINE-HYDROXYLASE ENZYME

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.