Affiliations: [a] Center for Research, Prevention, and Treatment of Atherosclerosis, Department of Medicine B, Hebrew University-Hadassah Medical School and Hadassah University Hospital, Jerusalem | [b] Department of Cardiology, Hebrew University-Hadassah Medical School and Hadassah University Hospital, Jerusalem | [c] Department of Human Genetics, Hebrew University-Hadassah Medical School and Hadassah University Hospital, Jerusalem
Correspondence:
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Corresponding author: Vardiella Meiner, M.D., Department of Human Genetics, Hadassah University Hospital, PO Box 12000, Jerusalem 91120 Israel. Tel.: +972 2 6777618; Fax: +972 2 6777499; E-mail: [email protected].
Abstract: Apolipoprotein E (apo E) is an essential constituent of several plasma lipoproteins, and plays an important role in lipoprotein metabolism. The apo E gene exhibits two common functional polymorphisms, producing 3 isoforms known to be associated with the risks of developing cardiovascular disease and susceptibility to Alzheimer's disease. Numerous different methods have been established for determining the three apo E isoforms, yet there are disadvantages and ambiguities associated with all of them. We used a method adapted for multiplex automated primer extension analysis by improving a commercially available protocol (SNaPshot™) and simultaneously typing apo E single nucleotide polymorphisms (SNPs) encoding for isoforms at codon 112 and 158. This protocol relies on the extension with fluorescent dideoxyNTPs of a primer that ends one nucleotide 5' of a given SNP (minisequencing). Improvement of the method is achieved by incorporating into the minisequencing reaction two pooled primers corresponding to both apo E SNPs followed by analysis on an ABI PRISM 310 DNA sequencer. We found full concordance with genotypes determined using universal heteroduplex. This method is readily available for many laboratories and is a simple, unequivocal easy to use technique suitable for large amount of clinical samples that may provide a significant improvement over previously reported methods for apo E genotyping.
Keywords: Apolipoprotein E, genotyping, single nucleotide polymorphism, Alzheimer's disease, coronary artery disease
