Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles.

Phenylketonuria (PKU, MIM 261600; EC 1.14.16.1) results from mutations in the phenylalanine hydroxylase (PAH) gene. Newborn metabolic disease screening uses blood dried on filter paper (DBS) to prospectively identify candidate newborns affected with PKU via an elevated concentration of phenylalanine. However, it is then important to confirm the specific category of PKU since classical PKU requires a stringent diet while milder categories may not require diet and a very important BH4-responsive category may be treated with the PAH cofactor 6R-tetrahydrobiopterin (BH4). Since there is a close genotype-phenotype correlation in PKU, determining the PAH genotype can be extremely important for therapy as well as prognosis. A simple and rapid method of accurately determining the PAH genotype would be a valuable addition to the diagnosis of PKU. Described herein is a means to identify variants in the PAH gene using high-resolution melt profiling, which compares the thermal denaturation profile of a patient sample to that of a control. Regions where the patient and control samples produce a common profile were not further evaluated, while those regions where the patient profile deviates from the control were assessed by DNA sequencing. Additionally described is a scheme utilizing redundant analysis with melt profile controls and a novel multiplex genotyping assay to triage deviation owing to known polymorphisms. Two mutations were identified in 93 of the 95 patients assessed and in the remaining two patients a single mutation was identified. Melt profiling provided 99% sensitivity to identify sequence variants in the PAH gene.