Rapid Identification of Apolipoprotein E Genotypes by Multiplex Amplification Refractory Mutation System PCR and Capillary Gel Electrophoresis

Apolipoprotein E (Apo E) genotyping on its own is neither sufficiently sensitive nor specific enough for use as a predictive diagnostic test for Alzheimer disease (AD) (1). Nevertheless, it is still of diagnostic value in the classification of type III hyperlipidemia (2). Furthermore, there exists a great research need and interest for Apo E genotyping in conjunction with other data relating to genetic polymorphisms. Several independent studies have now established that the inheritance of one or more Apo e4 alleles increases an individual’s risk of developing atherosclerosis and AD (3)(4). Conversely, the inheritance of one or more e2 alleles confers protection against AD (5) and is associated with lower lipid concentrations (6). Thus, a rapid and simple genotyping test is needed for any laboratory that takes part in clinical research related to Apo E.Apo E is a polymorphic protein consisting of a single polypeptide chain, 299 amino acids long. In plasma, it exists mainly in a nonglycosylated form. The three major isoforms of the protein are Apo E2, Apo E3, and Apo E4. These differ from each other by cysteine-arginine interchanges at amino acid residues 112 and 158. The biosynthesis of each protein isoform is under the control of three independent codominant alleles, e2, e3, and e4, located at a single Apo E gene locus on chromosome 19q13. Depending on the inheritance of any two alleles, six common Apo E genotypes are possible, three homozygotes (e2e2, e3e3, and e4e4) and three heterozygotes (e2e3, e3e4, and e2e4) (7).Currently, several different Apo E genotyping techniques have been described; these include minisequencing (8), single-strand conformation polymorphism (9), allele-specific oligonucleotide probes (10), oligonucleotide ligation assays (11), restriction isotyping with Hha I or Afl III/ Hae II (12)(13), and the amplification refractory mutation system (ARMS) (14). The ARMS …