Molecular heterogeneity of nonphenylketonuria hyperphenylalaninemia in 25 Danish patients.

Abstract

Phenylalanine hydroxylase (PAH; EC 1.4.16.1) is a liver-specific enzyme that catalyzes the initial and rate-limiting step in the catabolism of phenylalanine. The reaction is dependent on the cofactor tetrahydrobiopterin (BH[sub 4]) and results in the formation of tyrosine. Deficiency of PAH activity can result in a wide spectrum of metabolic and clinical phenotypes, depending on the degree of PAH depression. Thirty Danish children with non-PKU HPA were detected through the national neonatal screening program in the period 1967-1992. For this study, DNA samples from 25 of these patients were available. Criteria for diagnosis of non-PKU HPA include persistently elevated phenylalanine of 150-600 [mu]M and the ability to eliminate an oral phenylalanine load test dose of 0.1 g phenylalanine/kg body wt within 24 h. All 13 exons and adjoining splice signals of the PAH gene from the non-PKU HPA probands were amplified by PCR and scanned for the presence of mutations by DGGE according to previously described procedures. All samples displaying an latered electrophoretic band pattern were subjected to direct sequencing. A point mutation with potential influence on enzyme structure and/or function was identified on 47 of 50 chromosomes, and both mutant alleles were characterized in 23 of 25 probands. Formore » most of the identified mutations, the influence on enzyme activity has not yet been formally verified by in vitro expression. However, the fact that only two mutations were found in each patient by scanning the entire coding sequence and splice sites of the PAH gene provides substantial evidence that these mutations are indeed causative.« less