Integrated single-label liquid-phase assay of APOE codons 112 and 158 and a lipoprotein study in british women

Background: Apolipoprotein E (APOE) is an important element of lipid metabolism and, hence, cardiovascular disorders. APOE has 3 main allelic variants: e3, e4, and e2. Of these, ε3 is the most common, followed by e4 and e2. The associations of these isoforms with cardiovascular disorders and Alzheimer disease have been widely studied in different populations. Most of the genotyping in these studies has been performed with gel-based methods, which have important limitations, particularly for large epidemiologic studies. We therefore developed an integrated one-tube liquid-phase assay. Methods: To measure APOE isoforms, we developed an integrated single-label liquid-phase fluorescence assay containing 2 PCR primers, 2 probes, and 2 quencher oligonucleotides. We used a 384-well LightTyper, but the assay would be generically applicable for use with any fluorescence detector with thermal ramp control. We validated this method and applied it in the British Women's Heart and Health Study. Results: There were 4 melting peaks, at 41, 56, 61, and 69 °C, which generated 6 distinctive patterns representing genotypic combinations of e3, e4, and e2. The magnitude and direction of the associations found with total cholesterol, HDL-cholesterol, triglycerides, and estimated LDL-cholesterol were consistent with previous reports. Conclusion: The one-tube LightTyper assay presented here enables accurate, convenient, and economical genotyping of APOE and can be used for large epidemiologic studies.