Advancing Neonatal Screening for Pyridoxine-Dependent Epilepsy-ALDH7A1 Through Combined Analysis of 2-OPP,...

Pyridoxine-dependent epilepsy (PDE) represents a group of rare developmental and epileptic encephalopathies. The most common PDE is caused by biallelic pathogenic variants in ALDH7A1 (PDE-ALDH7A1; OMIM #266100), which encodes α-aminoadipate semialdehyde (α-AASA) dehydrogenase, a key enzyme in lysine catabolism. Affected individuals present with seizures unresponsive to conventional anticonvulsant medications but responsive to high-dose of pyridoxine (vitamin B6). Adjunctive lysine restriction and arginine supplementation have also shown potential in improving neurodevelopmental outcomes. Given the significant benefit of early intervention, PDE-ALDH7A1 is a strong candidate for newborn screening (NBS). However, traditional biomarkers are biochemically unstable at room temperature (α-AASA and piperideine-6-carboxylate) or lack sufficient specificity (pipecolate), limiting their utility for biomarker-based NBS. The recent identification of two novel and stable biomarkers, 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylate (2-OPP) and 6-oxo-pipecolate (oxo-PIP), offers renewed potential for biochemical NBS. We evaluated the feasibility of incorporating 2-OPP, oxo-PIP, and pipecolate into routine butylated FIA-MS/MS workflows used for biochemical NBS. A total of 9402 dried blood spots (DBS), including nine confirmed PDE-ALDH7A1 patients and 9393 anonymized controls were analyzed using a single multiplex assay. 2-OPP emerged as the most sensitive biomarker, identifying all PDE-ALDH7A1 patients with 100% sensitivity and a positive predictive value (PPV) of 18.4% using a threshold above the 99.5th percentile. Combining elevated 2-OPP (above the 99.5th percentile) with either pipecolate or oxo-PIP (above the 85.0th percentile) as secondary marker detected within the same multiplex FIA-MS/MS assay further improved the PPVs to 60% and 45%, respectively, while maintaining compatibility with butanol-derivatized method. Notably, increasing the 2-OPP threshold above the 99.89th percentile, in combination with either pipecolate or oxo-PIP above the 85.0th percentile resulted in both 100% sensitivity and 100% PPV. This study supports the strong potential of 2-OPP-based neonatal screening for PDE-ALDH7A1 within existing NBS infrastructures. The ability to multiplex 2-OPP, pipecolate and oxo-PIP within a single assay offers a robust, practical, high-throughput and cost-effective approach. These results support the inclusion of PDE-ALDH7A1 in existing biochemical NBS panels. Further prospective studies in larger cohorts are needed to refine cutoffs and confirm clinical performance.