RESUMO
Apolipoprotein E (apo E) polymorphism is associated with increased risk of cardiovascular and Alzheimer diseases, making its genotyping of potentially predictive value. We developed a rapid, reliable and specific method for determining APOE genotypes by fluorescent resonance energy transfer (FRET) over a high number of samples in a single run using a LightTyper device and dedicated probes. The method, validated with 75 blood samples, was designed to simultaneously detect three common APOE polymorphisms, epsilon(2,) epsilon(3) and epsilon(4), and to identify in a single reaction any of the six following genotypes: epsilon(2)/epsilon(2), epsilon(3)/epsilon(3), epsilon(4)/epsilon(4), epsilon(3)/epsilon(4), epsilon(4)/epsilon(2), epsilon(3)/epsilon(2). The assay involved three phases: (1) DNA extraction, (2) amplification, and (3) melting curve analysis using FRET technique. Briefly, genomic DNA of patients was extracted from total blood. Fragment of APOE was amplified by a first PCR run. Fluorescent labeled probes were added in a second PCR run. FRET genotyping showed following distribution: (1) 1.3% for epsilon(2)/epsilon(2) and epsilon(4)/epsilon(4) homozygotes, (2) 4.0, 6.6 and 14.7% for epsilon(2)/epsilon(4), epsilon(2)/epsilon(3) and epsilon(3)/epsilon(4) heterozygotes, respectively, and (3) 72.0% for epsilon(3)/epsilon(3) homozygotes. Moreover, a careful analysis of the FRET melting curves allowed us to determine the presence of a new polymorphism on the third position of the codon 158 (-AAGCGT-), namely, two nucleotides downstream from the known polymorphism. When the FRET analysis was compared to those obtained by RFLP and sequencing, the presence of this new polymorphism was confirmed only by sequencing thus indicating that RFLP analysis is not always reliable for genotyping.