A silent mutation induces exon skipping in the phenylalanine hydroxylase gene in phenylketonuria

An A-->T substitution in cDNA nucleotide 1197 (c.1197A/T) of the human phenylalanine hydroxylase (PAH) gene has been regarded as a silent mutation, because both the wild-type (GUA) and the mutant (GUU) alleles encode a valine residue at codon 399 (V399 V). The nucleotide c.1197 is located at the 3'-end of exon 11 at position -3 of the exon-intron junction. To explore whether the substitution exerts any effects on the processing of the PAH mRNA, illegitimate PAH transcripts from lymphoblast cultures of a phenylketonuria (PKU patient heterozygous for c.1197A/T were analyzed by the polymerase chain reaction following reverse-transcription (RT-PCR). mRNAs with an exon 11 deletion were revealed. Furthermore, by using an R408 W mutation in the paternal allele as a marker, sequence analysis of the RT-PCR products indicates that virtually all PAH transcripts from the maternal allele with the c.1197A/T substitution do not contain exon 11. To address whether this substitution is the main determinant for exon skipping, PAH minigenes with or without the substitution were constructed and transfected to a human hepatoma cell line. Analysis of the transcription products by S1 nuclease mapping clearly indicated that such exon 11 skipping was directly associated with the c.1197A/T substitution. Thus, this study demonstrates that the c.1197A/T substitution in the PAH gene is not just a neutral polymorphism but a mutation that induces post-transcriptional skipping of exon 11 leading to a PKU phenotype.