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
determined mainly by the Kringle IV size polymorphism. We have optimized
and characterized pulsed field gel electrophoresis (PFGE) for apo(a)
genotyping. METHODS: Established PFGE protocols were adjusted. The changes
included the following: (a) increased DNA yields by the use of all
leukocytes for isolation from either 3 mL of fresh EDTA whole blood or 250
microL of frozen buffy coats; (b) increased efficiency of Kpn1 digestion
by the inclusion of a digestion buffer wash; (c) reduction of assay time
by the use of capillary blotting; (d) 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 for 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 Kringle 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 technique by PFGE were improved, and accuracy and
reproducibility were preserved. The optimized procedure is promising for
apo(a) genotyping on frozen buffy coats from large epidemiological
studies