A new spectrum of phenylalanine hydroxylase gene variants in newborns with hyperphenylalaninaemia: results of the first two years of expanded neonatal screening in Russia

Background. Since 2023, the Russian Federation has implemented an expanded neonatal screening program, incorporating molecular genetic diagnostics for phenylketonuria (PKU)/ hyperphenylalaninemia (HFA) in newborns with elevated phenylalanine levels. The analysis of a large-scale group of patients collected from all regions of Russia will allow the identification and evaluation of a wide range of allelic variants of the PAH gene.

Methods. 1254 dried blood spots on filter paper and whole blood samples in EDTA tubes were collected from newborns who, during screening by tandem mass spectrometry (TMS), showed elevated levels of phenylalanine (>120 µmol/L), phenylalanine/tyrosine ratio (>3 µmol/L) on the 2nd day of life or later. A repeat analysis by TMS was performed for these newborns, alongside an analysis of 25 common variants using allele-specific MLPA (1252 probands). In cases where one pathogenic variant was identified in the PAH gene and/or repeated elevation of phenylalanine levels was observed, subsequent panel-based NGS sequencing (genes: PAH, PTS, QDPR, GCH1, PCBD1, SPR, DNAJC12) and quantitative MLPA analysis were conducted.

Results. Among 540 newborns (1080 chromosomes), two or more pathogenic variants in the PAH gene were identified. For five probands, two variants were detected in genes associated with the development of hyperphenylalaninemia (HPA). The frequency of molecularly confirmed PKU in the Russian Federation was 1:4501, which differs from the previously estimated frequency of 1:7000.

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