Newborn screening for pyridoxin dependent epilepsy by FIA-MS / MS: a feasibility study

Background Pyridoxine-dependent epilepsy (PDE) is a rare developmental and epileptic encephalopathy caused by biallelic pathogenic variants in ALDH7A1, which encodes α-aminoadipic semialdehyde (α-AASA) dehydrogenase, an enzyme involved in lysine catabolism. This disorder is characterized by seizures resistant to conventional antiseizure medications but responsive to pharmacological doses of vitamin B6. Additionally, lysine restriction and arginine supplementation have shown promise in improving neurodevelopmental outcomes. Without early diagnosis and treatment, affected individuals suffer from uncontrolled seizures, high morbidity, and even mortality. Despite being treatable, PDE is rarely diagnosed at birth, leading to delayed specific treatment, unnecessary antiseizure medication trials, recurrent hospitalizations and preventable neurological damage. Given that early intervention dramatically improves outcomes, PDE is an ideal candidate for newborn screening (NBS). However, traditional biochemical markers (α-AASA and P6C) are unstable at room temperature, limiting their utility in biomarker-based NBS programs. Pipecolic acid, though often elevated in affected individuals, lacks sufficient specificity as a standalone biomarker. The discovery of two novel, stable biomarkers (2-OPP and 6-oxopipecolic acid) has renewed interest in biochemical NBS for PDE. Methods This study aimed to incorporate these biomarkers for neonatal screening using multiplex FIA-MS / MS into the routine measurement of amino acids and acylcarnitines (butylated method). Residual dried blood spots (DBS) from five confirmed patients and 9349 control samples (fresh anonymized samples with negative results for the 23 diseases screened in our NBS program) were screened for 2-OPP, 6-oxopipecolic acid, P6C and pipecolic acid. Results Our findings suggest that 2-OPP is the most promising biomarker for PDE screening, with a sensitivity of 100 % and a specificity of 99.54 % when using a cutoff above the 99.5th percentile. The positive predictive value reached 100 % when increased 2-OPP was combined with an elevation in any of the three additional candidate biomarkers also measured in first-tier testing: pipecolic acid, 6-oxo-pipecolic acid or P6C. Conclusion These results strongly support the feasibility of implementing PDE screening in newborns, paving the way for earlier diagnosis and improved patient outcomes.