A simple and rapid polymerase chain reaction-based method for detecting a prevalent mutation (R413P) in Japanese phenylketonuria patients.

[classical phenylketonuria]

A convenient molecular method for the detection of R 413 P (1238 G-->C ) mutation in exon 12 of the phenylalanine hydroxylase gene , one of the prevalent mutations among Japanese patients with classical phenylketonuria (PKU ) is described . The mutation was previously detected by polymerase chain reaction (PCR )-direct sequencing or allele specific oligonucleotide hybridization . However , these methods were cumbersome and only a few laboratories could provide such a diagnostic service . An improved version of the method has been developed here , involving 30 cycles of PCR following restriction enzyme digestion . In the upstream primer encompassing G-1207 to C-1237 , two substitutions are artificially introduced , so that a Bam-HI site involving C-1238 is introduced in the copies of the mutant allele . With the use of this method , R 413 P -homozygote and -heterozygote can be readily and unequivocally distinguished from normal using genomic DNA extracted from peripheral blood leukocytes . Among 10 Japanese PKU patients investigated , three were homozygous and three were heterozygous for the R 413 P allele , whereas four did not carry this mutant allele , indicating that the prevalence of the mutant allele is 45 %. The result suggests that it is technically feasible to develop a program for carrier detection of the mutation in the Japanese population .