Single-step mutation scanning of the 6-pyruvoyltetrahydropterin synthase gene in patients with hyperphenylalaninemia

Background: Deficiency of 6-pyruvoyltetrahydropterin synthase (PTPS) is a r ecessively inherited disorder that leads to depletion of 5,6,7,8-tetrahydro biopterin, the obligatory cofactor for hydroxylation of phenylalanine, tyro sine, and tryptophan. A marker for neonatal detection of PTPS deficiency is hyperphenylalaninemia (HPA). Molecular analysis would provide a simple and reliable means for distinguishing PTPS deficiency from other potential cau ses of HPA. Methods: We developed a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) that rapidly scans the six coding seque nces and all splice sites of the PTPS gene (PTS) for mutations. This method was used to examine the status of the PTS gene in control samples with kno wn PTS mutations and in five patients with PTPS deficiency. Results: Two features of the PTS gene posed particular problems in relation to DGGE analysis: the very high GC content of exon 1, and a 15-bp poly(dT) stretch in the acceptor splice site of intron 1. Both problems were solved by special design of amplification primers. PCR and DGGE conditions were a djusted to allow simultaneous analysis of all six regions of the PTS gene. Using this one-step approach, all control mutations were readily resolved. Among the five PTPS patients, four mutations were identified, including IVS 1-3C-->G, IVS2-7T-->A, V57del, and V97M (289G-->A). The IVS1-3C-->G mutatio n was shown by reverse transcription-PCR analysis to produce multiple splic e variants. Conclusions: We have established a fast and reliable screening method for d etection of mutations and small deletions/insertions in the PTS gene. This method should be useful for rapid diagnosis of PTPS deficiency in newborns with HPA. (C) 1999 American Association for Clinical Chemistry.