3-O-methyldopa profiling in aromatic L-amino-acid decarboxylase deficiency: from diagnosis to follow-up under gene therapy using dried blood spots LC-MS/MS

Abstract Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare neurometabolic disorder requiring early and accurate diagnosis, especially with the implementation of gene therapy. Herein, we developed and thoroughly validated a rapid and reliable LC-MS/MS method for the quantification of 3-O-methyldopa (3-OMD) in dried blood spots (DBS), offering a robust, sensitive, and minimally invasive alternative to cerebrospinal fluid analysis for the diagnosis of AADC deficiency. Age-specific 3-OMD reference intervals and diagnostic cut-off values were defined to determine diagnostic decision thresholds for AADC deficiency in a pediatric population. The proposed upper cut-offs were 2.89 µmol/L for neonates, 1.03 µmol/L for children aged 28 days to 10 years, and 0.72 µmol/L for children older than 10 years. For the first time, we report longitudinal 3-OMD profiles in AADC patients undergoing gene therapy, offering preliminary insights into biomarker kinetics post-treatment. In patients treated with gene therapy, 3-OMD levels decreased over time but remained above the established reference ranges. Overall, the findings support the clinical implementation of blood-based 3-OMD quantification for diagnosis of AADC deficiency. This method could enable rapid diagnosis in the pediatric population presenting with neurological symptoms that are not always clearly indicative of AADC deficiency. Further research involving larger patient cohorts, extended follow-up periods, and integration of clinical outcome measures is warranted to better define the role of 3-OMD in therapeutic monitoring and to identify additional biomarkers that may further optimize therapeutic strategies.