Diagnóstico del déficit de acil-CoA deshidrogenasa de cadena media mediante la reacción en cadena por la polimerasa en tiempo real

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common hereditary defect of fatty acid oxidation in humans. A single A to G nucleotide transition at position 985 (nt c985 A-→G) of the MCAD gene, which results in the substitution of lysine by glutamic acid at residue 329 of the enzyme (K329E), represents more than 81% of alleles causing MCAD deficiency. PCR-based technologies are now being used widely for the identification of this mutation, however they involve multiple steps and are time-consuming. In this work we present a new detection method based on real-time PCR amplification coupled to fluorescence resonance energy transfer (FRET) to detect nt c985 A→G mutation of the MCAD gene. With this method we analyzed 18 individuals, previously diagnosed of MCAD deficiency by biochemical techniques, and 25 healthy controls. The results were consistent with those obtained by direct sequencing of PCR products. In conclusion, this new method combines simple sample processing and rapid analysis; it therefore affords both high-throughput genotyping and rapid results. These advantages could be applied for neonatal screening of nt c985 A→G mutation in MCAD gene in risk population.