PMID- 10585341
OWN - NLM
STAT- MEDLINE
DCOM- 19991217
LR  - 20190722
IS  - 0009-9147 (Print)
IS  - 0009-9147 (Linking)
VI  - 45
IP  - 12
DP  - 1999 Dec
TI  - Single-step mutation scanning of the 6-pyruvoyltetrahydropterin synthase gene in 
      patients with hyperphenylalaninemia.
PG  - 2102-8
AB  - BACKGROUND: Deficiency of 6-pyruvoyltetrahydropterin synthase (PTPS) is a
      recessively inherited disorder that leads to depletion of 5,6,7,
      8-tetrahydrobiopterin, the obligatory cofactor for hydroxylation of
      phenylalanine, tyrosine, 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
      causes of HPA. METHODS: We developed a method based on PCR in combination with
      denaturing gradient gel electrophoresis (DGGE) that rapidly scans the six coding 
      sequences 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 known 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 adjusted 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 IVS1-3C-->G, IVS2-7T-->A, V57del, and V97M (289G-->A). The IVS1-3C-->G 
      mutation was shown by reverse transcription-PCR analysis to produce multiple
      splice variants. CONCLUSIONS: We have established a fast and reliable screening
      method for detection 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.
FAU - Romstad, A
AU  - Romstad A
AD  - The John F. Kennedy Institute, Gl. Landevej 7, 2600 Glostrup, Denmark.
FAU - Guldberg, P
AU  - Guldberg P
FAU - Blau, N
AU  - Blau N
FAU - Guttler, F
AU  - Guttler F
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Clin Chem
JT  - Clinical chemistry
JID - 9421549
RN  - EC 4.6.- (Phosphorus-Oxygen Lyases)
RN  - EC 4.6.10 (6-pyruvoyltetrahydropterin synthase)
SB  - IM
MH  - Alleles
MH  - Electrophoresis, Gel, Pulsed-Field/methods
MH  - Exons
MH  - Genetic Testing/methods
MH  - Humans
MH  - *Mutation
MH  - Phenotype
MH  - Phenylketonurias/diagnosis/enzymology/*genetics
MH  - Phosphorus-Oxygen Lyases/*genetics
MH  - Polymerase Chain Reaction/methods
MH  - Temperature
EDAT- 1999/12/10 00:00
MHDA- 1999/12/10 00:01
CRDT- 1999/12/10 00:00
PHST- 1999/12/10 00:00 [pubmed]
PHST- 1999/12/10 00:01 [medline]
PHST- 1999/12/10 00:00 [entrez]
PST - ppublish
SO  - Clin Chem. 1999 Dec;45(12):2102-8.