CHARACTERIZATION OF THE GENE FOR APOLIPOPROTEIN E5-FRANKFURT (GLN(81)-]LYS, CYS(112)-]ARG) BY POLYMERASE CHAIN-REACTION, RESTRICTION ISOTYPING, AND TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

A new apolipoprotein (apo) E variant, apoE5-Frankfurt, was identified in a 43-year-old male with moderate hypercholesterolemia. On isoelectr ic focusing in an immobilized pH gradient, apoE5-Frankfurt migrated to a position more cathodic than apoE4 (Cys112 -> Arg). On sodium dodecy l sulfate-gel electrophoresis, its apparent molecular weight could not be distinguished from that of the three common apoE isoforms (E2, E3 and E4). Restriction isotyping with CfoI (HhaI) showed that apoE5-Fran kfurt had arginine in positions 112 and 158 of the mature protein, sug gesting that the mutation accounting for the additional positive charg e had occurred in an epsilon4 allele. The third and the fourth exon of the apoE gene were amplified using the polymerase chain reaction and analyzed by temperature gradient gel electrophoresis. This suggested t hat there were two mutations in the fourth exon of the mutant allele. Cloning and sequencing disclosed that, apart from the exchange of argi nine for cysteine in position 112, a C to A substitution replaced glut amine (CAA) in position 81 by lysine (AAA).