Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option
ABSTRACT PURPOSE: Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients with PDE, the disorder is caused by the deficient activity of the enzyme α-aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly from Spain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease. METHODS: The 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/MS and by sequencing analysis of messenger RNA (mRNA) and genomic DNA of ALDH7A1. KEY FINDINGS: Most of the patients had seizures in the neonatal period, but they responded to vitamin B6 administration. Three patients developed late-onset seizures, and most patients showed mild-to-moderate postnatal developmental delay. All patients had elevated PA and α-AASA levels, 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>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 and 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.
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ABSTRACT: Therapeutic options currently available for neonatal seizures are still unsatisfactory both in terms of efficacy and of risk for long-term neurotoxicity, even if there is growing recognition of their potential to worsen neurodevelopmental outcome. A recent paper by Slaughter and colleagues entitled "Pharmacological treatment of neonatal seizures: a systematic review" has been published with the aim to provide a treatment algorithm, but, due to the relative paucity of clinical studies, it relies mainly on traditional antiepileptic drugs and does not distinguish between different neonatal populations, especially preterm and hypothermic neonates, who might require a dedicated approach in order to improve seizure control and reduce side effects.Italian Journal of Pediatrics 06/2013; 39(1):37. DOI:10.1186/1824-7288-39-37 · 1.52 Impact Factor
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ABSTRACT: Our aim was to report two new cases of hyperlysinemia type I describing the clinical, biochemical and molecular features of the disease and the outcome of lysine restriction. Two children presented with febrile seizures followed by developmental delay, clumsiness and epilepsy. At age 2 and 8years a biochemical and genetic diagnosis of hyperlysinemia type I was confirmed and lysine-restricted diet was started in both cases. Three years after initiation of lysine restriction, case 1 had not suffered further seizures. In case 2, tremor and dysmetria improved, but fine motor clumsiness persisted. Mild cognitive impairment was present in both patients despite dietary treatment. Laboratory studies: Plasma, urine and cerebrospinal fluid amino acid concentrations were measured by ion exchange chromatography. Mutation analysis of the AASS gene was performed by directly sequencing the PCR products. The plasma lysine values were higher than 1200μmol/L in both cases. Additionally, an increase in dibasic aminoaciduria was observed. Lysine restriction decreased plasma lysine values and nearly normalised dibasic aminoaciduria. Mutational screening of the AASS gene revealed that case 1 was a compound heterozygote for c.2662+1_2662+5delGTAAGinsTT and c.874A>G and that case 2 was a compound heterozygote for c.976_977delCA and c.1925C>G. In conclusion, we present two children with hyperlysinemia type I and neurological impairment in which implementation of lysine-restricted diet achieved a mild improvement of symptoms but did not reverse cognitive impairment. The partial decrease of lysine concentrations and the normalisation of urine excretion of dibasic amino acids after lysine restriction further reinforce the possibility of this therapeutic intervention, although further investigations seem necessary.Molecular Genetics and Metabolism 07/2013; 110(3). DOI:10.1016/j.ymgme.2013.06.021 · 2.83 Impact Factor
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ABSTRACT: This paper describes a full detailed high performance liquid chromatography/tandem mass spectrometry method for the identification and quantification of human urine alpha-aminoadipic semialdehyde, biomarker of pyridoxine-dependent epilepsy. The ionization mode of the electrospray interface was negative and the metabolite was detected in the multiple reaction monitoring mode. Intra-day and inter-day laboratory precision were 4.64% and 7.30%, respectively, total run time was 3.5min. The calibration curve was linear between 0.25 and 10nmol with a correlation coefficient of the calibration line (R(2)≥0.9984); the limit of quantification was 0.25nmol within the control group. This simple, fast, high reproducible and robust procedure facilitates a rapid diagnosis of patients with pyridoxine-dependent epilepsy and can also be used to confirm the elevated urinary alpha-aminoadipic semialdehyde excretion in patients with other metabolic diseases as molybdenum cofactor and isolated sulphite oxidase deficiencies.Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 11/2013; 944C:141-143. DOI:10.1016/j.jchromb.2013.10.032 · 2.69 Impact Factor