title: Mutations in the phenylalanine hydroxylase gene: methods for their characterization.
abstract: Mutations in the phenylalanine hydroxylase (PAH) gene represent the root cause of PAH-deficient hyperphenylalaninemia. To date, more than 160 different mutations have been reported. Single-base substitutions and microdeletions account for the majority of molecular defects. This review provides a brief general introduction to various strategies for detection of PAH mutations, and summarizes our own methodological developments. We have established a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) for mutation scanning of the entire coding sequence and all exon/intron boundaries of the PAH. Systematic application of this method to the study of a large number of mutant chromosomes from hyperphenylalaninemic patients demonstrated a 98% diagnostic efficiency and a 100% mutation detection efficiency. We have created compromised PCR and DGGE conditions for simultaneous amplification and simultaneous mutation scanning of all PAH-coding fragments. This technique is convenient in a diagnostic setting and allows "same-day" DNA-based diagnosis of newborns with hyperphenylalaninemia. A further modification of the method allows unambiguous identification of known mutations, circumventing the cumbersome step of nucleotide sequencing.
authors.affiliation: Danish Center for Human Genome Research, John F Kennedy Institute, Glostrup.
authors.forename: P
authors.initials: P
authors.lastname: Guldberg
authors.forename: F
authors.initials: F
authors.lastname: Güttler
mesh_terms: D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D007231:Infant, Newborn; D009154:Mutation / Q000235:genetics*; D015997:Neonatal Screening / Q000379:methods; D010651:Phenylalanine Hydroxylase / Q000172:deficiency / Q000235:genetics*; D010661:Phenylketonurias / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D010651:Phenylalanine Hydroxylase