Project Summary: Pyridoxine-dependent epilepsy (PDE) is a developmental and epileptic encephalopathy that was historically defined by a positive clinical response to pyridoxine. Despite adequate seizure control, the majority of patients have developmental delay and intellectual disability. PDE is due to a deficiency of α-aminoadipic semialdehyde (α-AASA) dehydrogenase, a key enzyme in lysine metabolism that results in the accumulation of α-AASA and related metabolites. We initially focused on reducing the accumulation of α- AASA through a lysine-restricted diet and a competitive inhibitor of lysine transport. These novel treatment strategies improved the overall biochemistry and are associated with normal cognitive development. Although effective, treatment must be started early in life to prevent irreversible brain disease. Unfortunately, most patients are diagnosed after treatment is no longer effective. Our central hypothesis is that the delay in diagnosis and subsequent treatment is the main contributor of intellectual disability associated with this disease. The primary focus of this application is development of a newborn screen method for patients with PDE. Newborn screening provides an opportunity to diagnosis patients before irreversible neurologic damage occurs dramatically changing the natural history of this disease. Previous attempts at newborn screening were unsuccessful as α-AASA is unstable at room temperatures and, therefore, not practical for newborn screening. In our previous work, we identified a novel biomarker (6-oxo-pipecolate) that is stable at room temperatures and overcomes previous hurdles with newborn screening. We will demonstrate that newborns with PDE can be diagnosed at birth using current newborn screening techniques (Aim 1). These results will establish that 6-oxo-pipecoalte is elevated in affected newborns and will provide the basis for future studies focused on the sensitivity of our newborn screening method. Although α-AASA is an excellent diagnostic biomarker, the level of α-AASA does not correlate with disease severity or cognitive outcome. These results are confounded by the unstable nature of α-AASA, which makes it difficult to compare levels between samples and laboratories. We purpose that 6-oxo-pipecolate is an ideal biomarker to monitor treatment efficacy. We will evaluate the use of an at-home collection method for 6-oxo-pipecolate using our existing dried blood spot method. The benefit of at home treatment monitoring has never been more relevant than during this current pandemic due to the novel coronavirus COVID-19. We will demonstrate that measuring 6-oxo-pipecolate from dried blood spots collected at home is a feasible (Aim 2). These results will be critical for futures studies evaluating novel therapeutic approaches to this this treatable but severe neurologic disease.