An Oxford Nanopore Technologies–Based Sequencing Assay for Molecular Diagnosis of Phenylketonuria and Variant Frequencies in a Turkish Cohort

Background: Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutations in the PAH gene, resulting in deficient phenylalanine hydroxylase (PAH) enzyme activity and neurotoxic phenylalanine accumulation. Untreated PKU results in progressive neurodegeneration and severe intellectual disability. Neonatal screening has evolved from the Guthrie test to advanced techniques like HPLC, tandem mass spectrometry, and next-generation sequencing (NGS) for molecular confirmation. This study aimed to develop a rapid, scalable PAH genetic assay using Oxford Nanopore Technologies (ONTs) to enhance neonatal screening in high-prevalence regions like Türkiye, through accelerated, cost-effective genetic diagnostics.

Methods: An in-house panel was designed, implemented, and benchmarked against results obtained from the Illumina sequencing platform. A cohort of 40 PKU patients, previously diagnosed using Illumina platform, was selected for this study. Gene-specific primers were strategically designed to amplify exonic regions, untranslated segments, and exon–intron junctions of the PAH gene. Sequencing libraries were then prepared and processed using the MinION Mk1c instrument, with subsequent data analysis conducted through the Guppy software and complementary bioinformatics tools.

Results: The findings showed complete agreement between the ONT and Illumina platforms, corroborating the high fidelity and reliability of the ONT-based assay. All pathogenic variants previously identified through Illumina sequencing were accurately detected, albeit with varying observed allele frequencies. Notably, the most prevalent variants identified in the patient cohort were NC_000012.12(NM_000277.3):c.1066-11G > A with a frequency of 37.5% and NC_000012.12(NM_000277.3):c.782G > A, at 15%.

Conclusion: The ONT-based single-gene testing for PKU demonstrated complete concordance with Illumina sequencing, validating its accuracy and reliability. This method effectively detects pathogenic variants and offers a faster, cost-effective solution for neonatal screening, particularly beneficial in high-prevalence regions like Türkiye.