title: Rapid detection of the R408W and I65T mutations in phenylketonuria by glycosylase mediated polymorphism detection.
abstract: Mutation detection methods based upon chemical or enzymatic cleavage of DNA offer excellent detection efficiencies coupled with high throughput and low unit cost. We describe the application of the novel technique of Glycosylase Mediated Polymorphism Detection (GMPD) to the detection of two of the most common mutations of the PAH gene in the Irish population that cause phenylketonuria (PKU), R408W and I65T, which occur at relative frequencies of 41.0% and 10.4% respectively. GMPD assays for R408W and I65T were developed permitting fluorescent detection of cleavage products on the ALFexpresstrade mark automated DNA sequencer. The method was validated by screening a panel of PKU patients whose mutant genotypes had previously been characterised by standard methods. It also proved possible to perform multiplex detection of the two mutations by co-electrophoresis of GMPD products. GMPD is a rapid and robust method for the detection of the R408W and I65T mutations, whose key advantage lies in its use of a pair of enzymes with high cleavage efficiency to detect a number of mutations as compared to the use of individual digestions with a range of specific restriction endonuclease enzymes. Hum Mutat 17:432, 2001.
authors.affiliation: Department of Pathology, The Children's Hospital, Temple Street, Dublin 1, Republic of Ireland.
authors.forename: K A
authors.initials: KA
authors.lastname: O'Donnell
authors.forename: O
authors.initials: O
authors.lastname: Tighe
authors.forename: C
authors.initials: C
authors.lastname: O'Neill
authors.forename: E
authors.initials: E
authors.lastname: Naughten
authors.forename: P D
authors.initials: PD
authors.lastname: Mayne
authors.forename: T V
authors.initials: TV
authors.lastname: McCarthy
authors.forename: P
authors.initials: P
authors.lastname: Vaughan
authors.forename: D T
authors.initials: DT
authors.lastname: Croke
mesh_terms: D000483:Alleles; D001483:Base Sequence; D045647:DNA Glycosylases; D004252:DNA Mutational Analysis / Q000379:methods; D005091:Exons / Q000235:genetics; D005787:Gene Frequency; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D007494:Ireland; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000235:genetics*; D010661:Phenylketonurias / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D051981:Uracil-DNA Glycosidase
chemical_list: D010651:Phenylalanine Hydroxylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase