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Clinical Chemistry 52: 1420-1423, 2006. First published April 27, 2006; 10.1373/clinchem.2006.067082
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(Clinical Chemistry. 2006;52:1420-1423.)
© 2006 American Association for Clinical Chemistry, Inc.

Technical Briefs

Integrated Single-Label Liquid-Phase Assay of APOE Codons 112 and 158 and a Lipoprotein Study in British Women,

Mohammad Reza Abdollahi1,a, Philip A.I. Guthrie1, George Davey Smith2, Debbie A. Lawlor2, Shah Ebrahim3 and Ian N.M. Day1,4

1 Bristol Genetic Epidemiology Laboratory and 2 Department of Social Medicine, University of Bristol, Bristol, United Kingdom; 3 Department of Epidemiology & Population Health, London School of Hygiene & Tropical Medicine, London, United Kingdom; 4 Human Genetics Division, Southampton General Hospital, Southampton, United Kingdom;

aaddress correspondence to this author at: Bristol Genetic Epidemiology Laboratory, University of Bristol, No. 24 Tyndall Ave., Bristol, United Kingdom BS8 1TQ; fax 44-117-331-1729, e-mail r.abdollahi{at}bristol.ac.uk and rabdollahi{at}gmail.com


Abstract

Background: Apolipoprotein E (APOE) is an important element of lipid metabolism and, hence, cardiovascular disorders. APOE has 3 main allelic variants: {epsilon}3, {epsilon}4, and {epsilon}2. Of these, {epsilon}3 is the most common, followed by {epsilon}4 and {epsilon}2. 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 {epsilon}3, {epsilon}4, and {epsilon}2. 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.






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Copyright © 2006 by the American Association for Clinical Chemistry.