Comparison of apoE isoelectric focusing immunoblotting and apoE genotypes in young Japanese adults

Abstract

Common isoforms of apolipoprotein E (apoE) can be distinguished into apoE2, E3, and E4. Isoforms apoE2 and apoE4 are associated with dyslipidemia, while apoE4 is associated with the development of Alzheimer’s disease. ApoE mutation analysis is often performed solely through genetic testing. However, because charge-differentiated mutations frequently impact the biological activity of apoE, isoelectric focusing (IEF) serves as a critical method for protein charge analysis. The apoE phenotype determined by IEF immunoblotting, which allows simultaneous electrophoresis of 48 samples, was compared with apoE genotypes in 815 Japanese individuals. The results demonstrated a 97.6% match rate between apoE phenotype and genotype, with 795 of 815 cases matching. IEF immunoblotting revealed 3 apoE2/2, 577 E3/3, 11 E4/4, 61 E2/3, 14 E2/4, 130 E3/4, 3 E3/5, 1 E4/5, 1 E2/7, 12 E3/7, and 2 E4/7. Comparison with the restriction enzyme polymorphism assay using HhaI, which detects only genotypes ε2, ε3, and ε4, indicated that deviations were primarily due to apoE5 and E7, with one exception. One discordant case has phenotype E3/4 and genotype ε3/3. Sanger sequencing of the APOE gene identified a novel heterozygous APOE4 variant (Pro295Arg, p.Pro313Arg). This individual exhibited normolipidemia, with a serum total cholesterol level of 153 mg/dL, a high-density lipoprotein cholesterol level of 85 mg/dL, and a triglyceride level of 47 mg/dL. The IEF immunoblotting method, which permits simultaneous comparison of multiple samples, is a valuable technique for determining apoE phenotypes.