Detection and Identification of Base Alterations Within the Region of Factor V Leiden by Fluorescent Melting Curves

Background: Factor V Leiden (G1691A) is a common cause of inherited thrombosis. In fluorescent melting curve analysis, the Leiden mutation is distinguished from the wild-type by a decrease in melting temperature (Tm) of a wild-type probe. Because Tm depends on the type and position of the mismatch, other base alterations, such as the recently described base alteration A1692C, should be distinguishable from the true Leiden mutation. Methods and Results: Of 2,100 samples tested for the factor V Leiden mutation using a wild-type probe, 200 heterozygous or homozygous mutant samples were further tested using a Leiden probe. The Tm of the A1692C base alteration was 1.5 degrees C greater than the Leiden mutation with the wild-type probe and 8 degrees C less with the Leiden probe. One sample was heterozygous for a new base alteration G1689A with a Tm 0.8 degrees C greater than the Leiden mutation with the wild-type probe, and 10 degrees C less with the Leiden probe. Tm estimates from fluorescence melting curve analysis have intra-assay standard deviations of approximately 0.1 degrees C. Conclusions: Fluorescence melting curve analysis can distinguish between sequence alterations with Tms differing by less than 1 degrees C. This is the first demonstration of a widely applicable technique that can significantly increase the specificity of hybridization techniques without the need for sequencing.