RESUMO
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.
Assuntos
Epilepsia/tratamento farmacológico , Epilepsia/genética , Terapia Genética/métodos , Oligonucleotídeos Antissenso/uso terapêutico , Deficiência de Vitamina B 6/complicações , Aldeído Desidrogenase/genética , Linhagem Celular , Análise Mutacional de DNA , Epilepsia/etiologia , Éxons/genética , Feminino , Humanos , Hiperlisinemias/urina , Lactente , Recém-Nascido , Linfócitos/efeitos dos fármacos , Masculino , Mutação/genética , Polimorfismo de Nucleotídeo Único , Splicing de RNA , Sacaropina Desidrogenases/deficiência , Sacaropina Desidrogenases/urina , Espectrometria de Massas em TandemAssuntos
Transtornos Congênitos do Transporte de Aminoácidos/genética , Encefalopatias Metabólicas Congênitas/genética , Proteínas do Tecido Nervoso/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Transtornos Congênitos do Transporte de Aminoácidos/metabolismo , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Encefalopatias Metabólicas Congênitas/metabolismo , Pré-Escolar , Creatina/metabolismo , Análise Mutacional de DNA/métodos , Humanos , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Masculino , Trítio/metabolismoRESUMO
This paper reports a new, high-performance liquid chromatography/tandem mass spectrometry method for the separation and identification of human plasma short-chain acylcarnitine isomers. This simple, rapid procedure involves the use of a single sample previously shown to contain elevated acylcarnitine concentrations by flow injection analysis, and can separate two C4, three C5, two C5:1 and four C5-OH acylcarnitine isomers, thus permitting the differential diagnosis of certain fatty acid oxidation defects and organic acidemias.
