Аннотация
Introduction.Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine metabolism caused by pathogenic variants of the PAH gene. Allelic heterogeneity of the gene and the need for differential diagnosis of forms of hyperphenylalaninemia require molecular confirmation. Sanger sequencing remains valuable as an accessible and reliable method for variant verification and family analysis.The objectiveof the study was to develop and validate a unified protocol for PCR and Sanger sequencing of all coding exons of PAH with extended coverage of exon -intron boundaries.Materials and methods. A panel of 31 primers was developed to amplify 13 PAH exons, including overlapping amplicons for exon 13. Standardized PCR conditions (60 °C) were used. Validation was performed on five DNA samples, including a family trio. Sequencing was performed on an Applied Biosystems 3730xl.Results. The developed primer panel ensured reproducible and highly specific amplification of all target regions of the expected size. Sequencingdemonstrated high-quality chromatograms and complete coverage of the analyzed fragments, including overlapping regions. In the family trio, two pathogenic missense variants of the PAH gene in a state of compound heterozygosity were identified in the proband: c.728G>A (p.Arg243Gln) in exon 7 and c.1238G>C (p.Arg413Pro) in exon 12. Family analysis showed that the c.728G>A variant was paternally inherited, and c.1238G>C was maternally inherited. Additionally, a synonymous substitution c.735G>A was identified in the paternal allele. The established genotype fully corresponded to the clinical diagnosis of classical PKU and confirmed the diagnostic suitability of the developed approach.Conclusions. The proposed protocol is a reproducible, accessible and clinically informative method for molecular diagnostics of PKU and can be used both as a standalone tool and for validation of results obtained by other approaches.Keywords: phenylketonuria, hyperphenylalaninemia, exome sequencing, DNA mutation analysis.