Nonsense mediated mRNA decay affects nonsense transcripts levelsand in vitro response to gentamicin and ataluren in X-ALD

Abstract

Background: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene, characterized by increased concentrations of very long-chain fatty acids due to a defect in peroxisomal β-oxidation. Aminoglycosides and PTC124 can readthrough premature termination codons (PTCs), allowing the translation of full length proteins. Response to drugs found only in patients with the higher level of mRNA. Nonsense-mediated mRNA decay (NMD) is a mechanism, which degrades transcripts carrying PTCs and has an important role in response to treatments to promote readthrough. UPF1 RNA helicase are involved in this pathway. Objetives: Prove aminoglycosides and PTC124 in fibroblast cultures from patients with X-ALD. Analyze NMD efficiency in X-ALD fibroblasts. Materials and Methods: Fibroblasts from patients (p.Trp137*, p.Ser290*, p.Arg464*) were treated with different doses of gentamicin and PTC124. Protein expression was analyzed by Western blot. NMD was directly inhibited by using siRNA against UPF1 and indirectly by Cicloheximide. Levels of mRNA were determined by qPCR. * The study was approved by Comité Institucional de Etica para la Investigación Clínica (CIEIS) Polo Hospitalario, Provincia de Córdoba. * This work was supported by Fundación Florencio Fiorini , SECyT-UNC, PICT-FONCYT