Fluorescence-based, nonradioactive method for efficient detection of the pentanucleotide repeat (TTTTA)(n) polymorphism in the apolipoprotein(a) gene

Background: The apolipoprotein(a) [apo(a)] gene is a major predictor of pla sma lipoprotein(a) concentrations, an independent risk factor for cardiovas cular disease. The apo(a) gene contains a pentanucleotide repeat (PNR) poly morphism, 1.4 kb upstream from the apo(a) gene reading frame. This polymorp hism has been suggested to be important in control of apo(a) gene expressio n. Methods: We developed a fluorescence-based, nonradioactive procedure to det ect the PNR polymorphism. After amplification of the polymorphism by PCR, t he respective PCR products were separated by denaturing polyacrylamide gel electrophoresis and detected using a 3'-end fluorescently labeled oligonucl eotide as a probe. We used the method to characterize the PNR polymorphism pattern in 313 individuals, 195 Caucasians and 118 African Americans. The n ew method efficiently separated DNAs corresponding to the different PNR rep eats. Results: Among both ethnic groups, alleles containing eight PNRs were most common. Smaller PNRs were more common among African Americans, and larger P NRs were more common among Caucasians. Conclusions: We developed a nonradioactive technique that separates the PNR polymorphism in the apo(a) gene and can be used in other studies involving closely sized polymorphisms. (C) 2001 American Association for Clinical Ch emistry.