A rapid and cost-effective method for genotyping apolipoprotein E gene polymorphism.

BACKGROUND: Apolipoprotein E (ApoE) is a major cholesterol carrier and plays an important role in maintaining lipid homeostasis both in the periphery and brain. Human APOE gene is polymorphic at two single nucleotides (rs429358 and rs7412) resulting in three different alleles (ε2, ε3 and ε4). ApoE isoforms modulate the risk for a variety of vascular and neurodegenerative diseases; thus, APOE genotyping is crucial for predicting disease risk and designing individualized therapy based on APOE genotype. RESULTS: We have developed an APOE genotyping method that is based on allele-specific PCR methodology adapted to Real Time PCR monitored by TaqMan probe. Rather than using TaqMan probes specific for the two polymorphic sites, only one TaqMan probe is used as the polymorphic alleles are recognized by site-specific PCR primers. Each genotyping assay can be completed within 90 minutes and is applicable to high-throughput analysis. Using this protocol, we genotyped a total of 1158 human DNA samples and obtained a 100% concordance with the APOE genotype determined by sequencing analysis. CONCLUSION: The APOE genotyping assay we have developed is accurate and cost-effective. In addition, our assay can readily be applied to genotyping large sample numbers. Therefore, our APOE genotyping method can be used for assessing the risk for a variety of vascular and neurodegenerative diseases that have been reported to be associated with APOE polymorphism.

Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.