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: epsilon3, epsilon4, and epsilon2. Of these, epsilon3 is the most common, followed by epsilon4 and epsilon2. 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 degrees C, which generated 6 distinctive patterns representing genotypic combinations of epsilon3, epsilon4, and epsilon2. The magnitude and direction of the associations found with total cholesterol, HDL-cholesterol, triglycerides, and estimated LDL-cholesterol were consistent with previous reports.


CONCLUSIONS
The one-tube LightTyper assay presented here enables accurate, convenient, and economical genotyping of APOE and can be used for large epidemiologic studies.
Abdollahi
Mohammad Reza
MR
Bristol Genetic Epidemiology Laboratory and Department of Social Medicine, University of Bristol, Bristol, United Kingdom. r.abdollahi@bristol.ac.uk
Guthrie
Philip A I
PA
Smith
George Davey
GD
Lawlor
Debbie A
DA
Ebrahim
Shah
S
Day
Ian N M
IN
D000368:Aged; D001057:Apolipoproteins E / Q000235:genetics*; D002784:Cholesterol / Q000097:blood; D008076:Cholesterol, HDL / Q000097:blood; D008078:Cholesterol, LDL / Q000097:blood; D003062:Codon; D005260:Female; D005838:Genotype; D006801:Humans; D008074:Lipoproteins / Q000097:blood*; D008875:Middle Aged; D020033:Protein Isoforms / Q000235:genetics; D014280:Triglycerides / Q000097:blood; D006113:United Kingdom
D001057:Apolipoproteins E; D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D003062:Codon; D008074:Lipoproteins; D020033:Protein Isoforms; D014280:Triglycerides; D002784:Cholesterol