MUTATIONS IN THE PHENYLALANINE-HYDROXYLASE GENE - METHODS FOR THEIR CHARACTERIZATION

Mutations in the phenylalanine hydroxylase (PAH) gene represent the ro ot cause of PAH-deficient hyperphenylalaninemia. To date, more than 16 0 different mutations have been reported. Single-base substitutions an d microdeletions account for the majority of molecular defects. This r eview provides a brief general introduction to various strategies for detection of PAH mutations, and summarizes our own methodological deve lopments. We have established a method based on PCR in combination wit h denaturing gradient gel electrophoresis (DGGE) for mutation scanning of the entire coding sequence and all exon/intron boundaries of the P AH. Systematic application of this method to the study of a large numb er of mutant chromosomes from hyperphenylalaninemic patients demonstra ted a 98% diagnostic efficiency and a 100% mutation detection efficien cy. We have created compromised PCR and DGGE conditions for simultaneo us amplification and simultaneous mutation scanning of all PAH-coding fragments. This technique is convenient in a diagnostic setting and al lows ''same-day'' DNA-based diagnosis of newborns with hyperphenylalan inemia. A further modification of the method allows unambiguous identi fication of known mutations, circumventing the cumbersome step of nucl eotide sequencing.