Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option

<edited for length> PURPOSE: Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In most patients with PDE, the disorder is caused by deficient activity of the enzyme α-aminoadipic semialdehyde dehydrogenase (antiquitin protein), encoded by the ALDH7A1 gene. Our aim was the clinical, biochemical, & genetic analysis of 12 unrelated patients, mostly from Spain, to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease. METHODS: The disease was confirmed by the presence of α-aminoadipic semialdehyde (α-AASA) in urine measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) & pipecolic acid (PA) in plasma and/or cerebrospinal fluid measured by high performance liquid chromatography (HPLC)/MS/MS and by sequencing analysis of messenger RNA and genomic DNA of ALDH7A1. KEY FINDINGS: Most of the patients had neonatal seizures but they responded to vitamin B6. Three patients developed late-onset seizures, and most patients showed mild-to-moderate postnatal developmental delay. All patients had elevated PA & α-AASA levels, even those who had several years of pyridoxine treatment. 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, & psychomotor retardation. The mutational spectrum of the present patients included 12 mutations, five already reported (c.500A>G, c.919C>T, c.1429G>C c.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 & ex vivo splicing analysis showed the silent nucleotide change c.75C>T to be a novel splicing mutation creating a new donor splice site inside exon 1. Antisense therapy of the aberrant mRNA splicing in a lymphoblast cell line harboring mutation c.75C>T was successful. SIGNIFICANCE: The present results broaden our knowledge of PDE, provide information regarding the genetic background of PDE in Spain, afford data of use when making molecular-based prenatal diagnosis, and provide a cellular proof-of concept for antisense therapy application.