Exon deletions of the PAH gene in Italian hyperphenylalaninemics

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods such as Denaturing Gradient Gel Electrophoresis (DGGE), Denaturing High Performance Liquid Chromatography (DHPLC), direct sequencing. In recent years, it has been shown that a significant proportion of these undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes by gene-scanning methods due to the masking effect by the non-deleted allele. To date no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or Mild hyperphenylalaninemia. We have used Multiplex Ligation-dependent Probe Amplification (MLPA), Comparative Multiplex Dosage Analysis (CMDA) and Real- Time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian HPA patients. Overall, 4 deletions removing different Phenylalanine Hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving Exons 4-5-6-7-8 (systematic name c.353-?_912+?del) and Exon 6 (systematic name c.510-?_706+?del) have not been reported previously. In this study we show that exon deletion of the PAH gene account for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics. A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods such as Denaturing Gradient Gel Electrophoresis (DGGE), Denaturing High Performance Liquid Chromatography (DHPLC), direct sequencing. In recent years, it has been shown that a significant proportion of these undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes by gene-scanning methods due to the masking effect by the non-deleted allele. To date no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or Mild hyperphenylalaninemia. We have used Multiplex Ligation-dependent Probe Amplification (MLPA), Comparative Multiplex Dosage Analysis (CMDA) and Real- Time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian HPA patients. Overall, 4 deletions removing different Phenylalanine Hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving Exons 4-5-6-7-8 (systematic name c.353-?_912+?del) and Exon 6 (systematic name c.510-?_706+?del) have not been reported previously. In this study we show that exon deletion of the PAH gene account for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.