title: Identification and characterization of large deletions in the phenylalanine hydroxylase (PAH) gene by MLPA: evidence for both homologous and non-homologous mechanisms of rearrangement.
abstract: Large gene deletions and duplications were analyzed in 59 unrelated phenylketonuria (PKU) patients negative for phenylalanine hydroxylase (PAH) mutations on one or both alleles from previous exon by exon analysis. Using the novel multiplex ligation-dependent probe amplification (MLPA) method, a total of 31 partial PAH deletions involving single exons were identified in 31 PKU patients. Nineteen cases exhibited deletion of exon 5, and 12 cases provided evidence for the deletion of exon 3. Subsequently, using restriction enzyme digestion and DNA sequencing, three different large deletions, EX3del4765 (12 cases), EX5del955 (2 cases) and EX5del4232ins268 (17 cases) were identified and confirmed by long-range PCR and by the analysis of aberrant transcripts. Altogether, the 31 large deletions presented account for 3% of all PAH mutant alleles investigated in Czech PKU patients. Bioinformatic analysis of three breakpoints showed that the mutation EX3del4765 had arisen through an Alu-Alu homologous recombination, whereas two other mutations-the EX5del955 and EX5del4232ins268, had been created by a non-homologous end joining (NHEJ). We conclude that MLPA is a convenient, rapid and reliable method for detection of intragenic deletions in the PAH gene and that a relatively high number of alleles with large deletions are present in the Slavic PKU population.
authors.affiliation: Center of Molecular Biology and Gene Therapy, University Hospital Brno, Czech Republic. librokoz@hotmail.com
authors.forename: Libor
authors.initials: L
authors.lastname: Kozak
authors.forename: Eva
authors.initials: E
authors.lastname: Hrabincova
authors.forename: Jaromir
authors.initials: J
authors.lastname: Kintr
authors.forename: Ondrej
authors.initials: O
authors.lastname: Horky
authors.forename: Petra
authors.initials: P
authors.lastname: Zapletalova
authors.forename: Ivona
authors.initials: I
authors.lastname: Blahakova
authors.forename: Pavel
authors.initials: P
authors.lastname: Mejstrik
authors.forename: Dagmar
authors.initials: D
authors.lastname: Prochazkova
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D005091:Exons; D006801:Humans; D007438:Introns; D022521:Ligase Chain Reaction / Q000379:methods*; D008969:Molecular Sequence Data; D010651:Phenylalanine Hydroxylase / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion
chemical_list: D010651:Phenylalanine Hydroxylase