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

A convenient molecular method for the detection of R413P (1238G-->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, R413P-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 R413P 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.