SummaryPurposePyridoxine‐dependent epilepsy seizure (PDE;OMIM266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients withPDE, the disorder is caused by the deficient activity of the enzyme α‐aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by theALDH7A1gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly fromSpain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease.MethodsThe disease was confirmed based on the presence of α‐aminoadipic semialdehyde (α‐AASA) in urine measured by liquid chromatography tandem mass spectrometry (LC‐MS/MS) and pipecolic acid (PA) in plasma and/or cerebrospinal fluid (CSF) measured by high performance liquid chromatography (HPLC)/MS/MSand by sequencing analysis of messenger RNA (mRNA) and genomicDNAofALDH7A1.Key FindingsMost of the patients had seizures in the neonatal period, but they responded to vitaminB6 administration. Three patients developed late‐onset seizures, and most patients showed mild‐to‐moderate postnatal developmental delay. All patients had elevatedPAand α‐AASAlevels, even those who had undergone pyridoxine treatment for several years. The clinical spectrum of our patients is not limited to seizures but many of them show associated neurologic dysfunctions such as muscle tone alterations, irritability, and psychomotor retardation. The mutational spectrum of the present patients included 12 mutations, five already reported (c.500A>G, c.919C>T, c.1429G>Cc.1217_1218delAT, and c.1482‐1G>T) and seven novel sequence changes (c.75C>T, c.319G>T, c.554_555delAA, c.757C>T, c.787 + 1G>T, c.1474T>C, c.1093‐?_1620+?). Only one mutation, p.G477R(c.1429G>C), was recurrent; this was detected in four different alleles. Transcriptional profile analysis of one patient's lymphoblasts and ex vivo splicing analysis showed the silent nucleotide change c.75C>Tto be a novel splicing mutation creating a new donor splice site inside exon 1. Antisense therapy of the aberrantmRNAsplicing in a lymphoblast cell line harboring mutation c.75C>Twas successful.SignificanceThe present results broaden our knowledge ofPDE, provide information regarding the genetic background ofPDEinSpain, afford data of use when making molecular‐based prenatal diagnosis, and provide a cellular proof‐of concept for antisense therapy application.