Analysis by illegitimate transcription of a mutation in the 5{prime} splice site in exon 8 of the PAH gene

Up to now, 12 splice defects have been described within the PAH gene. Using PCR-SSCP and sequence analysis we have found a point mutation involving the last nucleotide in exon 8 (CAG/CAA). The G to A substitution does not alter the amino acid (Q204Q), but it may cause a splice defect, as it is included in the 5{prime} splice donor site, and the G at this position is highly conserved (80%) in all eukaryotic genes. We have analyzed by illegitimate transcription the PAH mRNA in lymphocytes of a patient bearing the mutation in a heterozygous fashion. After RT-PCR we observed once the appearance of an extra larger band, which could be due to the use of a cryptic splice site instead of the mutated one. Furthermore, sequencing of 6 clones of the band of expected size in the patient revealed that all had the normal sequence, in spite of the G to A substitution being found in the genomic DNA. In view of these results, we believe that the larger extra band represents the allele with the mutation which causes a highly unstable mis-spliced RNA. This splice defect could be, therefore, the disease causing mutation in the patient.