Optimization of apolipoprotein(a) genotyping with pulsed field gel electrophoresis

Background: Increased lipoprotein(a) is a risk factor for atherosclerosis, and its concentration in serum is inversely correlated with the size of the apoliprotein(a) [apo(a)] component. The size of the apo(a) gene is determi ned mainly by the Kringle IV size polymorphism. We have optimized and chara cterized pulsed field gel electrophoresis (PFGE) for apo(a) genotyping. Met hods: Established PFGE protocols were adjusted. The changes included the fo llowing: (a) increased DNA yields by the use of all leukocytes for isolatio n from either 3 mL of fresh EDTA whole blood or 250 mu L Of frozen buffy co ats; (b) increased efficiency of Kpn1 digestion by the inclusion of a diges tion buffer wash; (c) reduction of assay time by the use of capillary blott ing; cn, increased sensitivity by the use of four digoxigenin-labeled apo(a ) probes; and (e) identification using a single film by the inclusion of a digoxigenin-labeled lambda marker probe in addition to apo(a) probes in the hybridization mix. Results: In older Caucasians, 93% (buffy coats, n=468) were heterozygous fo r apo(a) gene size. An inverse correlation between serum lipoprotein(a) and the sum of Kringle IV alleles was found (y = -23x + 1553; r = -0.442; n = 468). Gel-to-gel variation was minimal (3%). Imprecision (SD) was one Kring le IV repeat (control sample containing eight fragments of 72-233 kb; n=34 electrophoretic runs). Conclusions: The practicality and sensitivity of the apo(a) genotyping tech nique by PFGE were improved, and accuracy and reproducibility were preserve d. The optimized procedure is promising for apo(a) genotyping on frozen buf fy coats from large epidemiological studies. (C) 1999 American Association for Clinical Chemistry.