Identification and characterization of a novel liver-specific enhancer of the human phenylalanine hydroxylase gene

The liver-specific phenylalanine hydroxylase catalyzes the conversion of phenylalanine to tyrosine. Genetic defects in the gene result in the autosomal recessive disorder phenylketonuria. We have identified a phenylalanine hydroxylase mutation, designated as -4173_-407del, in a hyperphenylalaninemic patient with a 3.7-kb deletion in the 5'-flanking region of the phenylalanine hydroxylase gene. Characterization of the deleted sequence has led to the identification of a novel liver-specific DNase I hypersensitive site located 3.3 kb upstream of the RNA initiation site of the phenylalanine hydroxylase gene. We show that this site comprises a liver-specific enhancer with cAMP responsiveness. We further show by mutational analysis that the enhancer carries a major hepatocyte nuclear factor-1-binding site important for the enhancer function but not for cAMP responsiveness. In transient transfection assays with a reporter gene, we demonstrate that a phenylalanine hydroxylase plasmid construct carrying the -4173_-407del mutation is severely impaired in phenylalanine hydroxylase transcriptional activity. Our data indicate that the 3.7-kb deletion uncovered in the genomic DNA of the phenylketonuria proband is linked to the observed phenylketonuria phenotype as a result of a deletion of the newly identified liver-specific enhancer. Our systematic approach to the analysis and subsequent discovery of the novel deletion mutation may be applicable to the search for other mutations in the 5' regulatory region of phenylalanine hydroxylase and other genes.