Characterization of the mouse phenylalanine hydroxylase mutation Pah(enu3)

Phenylketonuria (PKU) is an inborn error of metabolism that is inherited in an autosomal recessive manner. It arises from a deficiency of phenylalanin e hydroxylase, which is responsible for converting phenylalanine to tyrosin e and thereby hastening its catabolism. To produce mouse models for the stu dy of PKU, male mice were mutagenized with ethylnitrosourea and their proge ny were screened for the elevated phenylalanine levels characteristic of ph enylalanine hydroxylase deficiency. Of three mutant alleles recovered, two (Pah(enu1) and Pah(enu2)) were characterized previously and shown to be mis sense mutations. Sequencing of phenylalanine hydroxylase cDNA from the thir d mutant allele, Pah(enu3) revealed that two differently sized transcripts were being produced. These transcripts contained either a B-nucleotide inse rtion or a 5-nucleotide deletion and both of these modifications occurred a t the same location, the exon 11-exon 12 junction. Sequencing of the exon 1 1-intron 11 boundary revealed a T --> G transversion in the invariant GT di nucleotide of the wild-type 5' splice donor site. The analogous human Pah m utation would be called c.1199 + 2T > G. Sequence analysis also revealed tw o cryptic splice donor sites, upstream and downstream of the wild-type spli ce site, that appear to be used when the wild type is ablated and to thereb y yield the observed differently sized transcripts. The 5-nucleotide insert ion and the 5-nucleotide deletion are both predicted to cause frame shiftin g in exon 12 and exon 13, leading to premature termination. (C) 2001 Academ ic Press.