Simple Sequence-specific-Primer-PCR Method To Identify the Three Main Apolipoprotein E Haplotypes

Apolipoprotein E (ApoE) is involved in the binding, internalization, and catabolism of lipoprotein particles. ApoE is secreted by macrophages and hepatocytes and serves as a ligand for the LDL (Apo B/E) receptor and on hepatic tissues for the specific ApoE type 1 and 2 receptors for triglyceride-rich remnants (1)(2). The gene coding for ApoE is located on chromosome 19q13.32 and contains several polymorphic loci. The three common isoforms of ApoE, termed E2, E3, and E4, are defined by two single-nucleotide polymorphisms (SNPs) at positions 2059(T/C) and 2197(C/T) of the gene, which are located in the codons that code for amino acids 112 and 158 of the mature protein. The presence of a T allele at position 2059 (2059-T) of the gene defines a cysteine at position 112 (Cys112), whereas 2059-C defines an arginine for the same position (Arg112). Similarly, 2197-C on the gene defines an arginine at position 158 (Arg158), whereas 2197-T codes for cysteine for the same position (Cys158). ApoE3 (Cys112/Arg158) is the most common of the three isoforms found. The ApoE2 isoform Cys112/1Cys158 is associated with remnant hyperlipidemia because this isoform has a lower affinity for ApoE receptors. The ApoE4 (Arg112/Arg158) isoform is associated with increased hepatic synthesis of VLDL and is a risk factor for Alzheimer disease and other neurologic diseases. Other rare isoforms are also found and are associated with mixed hyperlipidemia (1). Several methods have been used for genotyping the three major ApoE isoforms. The problems inherent in phenotyping methods have led to the gradual adoption of genotyping methods for determination of ApoE isoforms in most laboratories (3). By far the commonest method has been PCR amplification followed by restriction fragment length polymorphism (RFLP) analysis. Although many variations of this methodology have been published, the original method used HhaI restriction endonuclease, which cleaves at the GCGC sequence that encodes Arg112 (E4) and Arg158 (E3, E4), but does not cut at the GTGC sequence that encodes Cys112 (E2 and E3) and Cys158 (E2) (4). Other methodologies used include capillary electrophoretic detection (5), Amplification Refractory Mutation System PCR (ARMS-PCR) (6)(7), and “real-time” PCR detection (8)(9). The PCR-RFLP mapping approach involves a single PCR reaction followed by restriction enzymatic digestion and genotypic assignment after subsequent analyses by electrophoresis. Although the methodology requires only a single PCR reaction per sample, the restriction enzyme digestion step increases the post-PCR processing time by 2 h. Furthermore, because incomplete restriction enzyme digestion can lead to false genotyping data, many researchers prefer to perform overnight digestions, thus increasing the assay time even further. In addition, the small size fragments produced after restriction enzyme digestion are usually resolved by acrylamide gels electrophoresis, thus limiting the number of samples that can be examined at one time. The real-time PCR and capillary electrophoretic detection methods are faster but require expensive dedicated instrumentation. The several ARMS-PCR methodologies reported identify the individual alleles by amplification using specific primers in combination with a common primer either in a four- (6) or two-PCR (7) reaction set-up, with the latter requiring denaturing acrylamide gel electrophoresis or capillary electrophoresis to eliminate amplification artifacts. The fact that the reported ARMS-PCR methodologies do not establish experimentally the cis/trans chromosomal orientation of the alleles at SNP positions 2059/2197 makes identification of the ApoE isoforms rather tedious and open to interpretational errors. We describe here an ApoE haplotype-specific sequence-specific-primer (SSP)-PCR methodology that identifies in three PCR reactions the allelic haplotypes that determine the three main ApoE isoforms. We have previously used this approach to determine the cis/trans chromosomal orientation of the individual nucleotides in SNPs located in the interleukin-13 gene and, thus, the specific allelic haplotypes of that gene (10). We designed two forward primers with variations in their 3′ nucleotides such that each was specific for one of the two variants in the 2059 locus and two reverse primers whose 3′-end nucleotides identified either of the nucleotide variants in the 2197 locus (MDL PRIMERS 1–4; Table 1 ). The sequence-specific forward and reverse primers were then combined in three haplotype-detecting reaction mixtures “Primer Mix E2” (primers 1 and 3), “Primer Mix E3” (primers 1 and 2), and “Primer Mix E4” (primers 2 and 4). Because this genotyping system is based on the presence or absence of PCR amplification by allele-specific primers, it is imperative to ensure PCR amplification for those reactions that do not produce haplotype-specific amplicons. For this reason, each ApoE-specific Primer Mix also contained a pair of “control primers” (primers 8 and 9), which amplify two regions of chromosome 6 in the HLA-DR locus, to verify PCR amplification in the absence of haplotype-specific amplification in each PCR reaction. Primers required for ApoE haplotype detection by SSP-PCR analysis. Primers required for ApoE haplotype detection by SSP-PCR analysis. For each DNA sample, 5 μL of each Primer Mix (E2, E3, and E4) was placed in the bottom of three 0.2-mL wells in a 96-well plate. To this we added a mixture of 8 μL of DNA (resuspended in DNase-free water or Tris buffer) and PCR reaction mixture containing PCR buffer [(NH4)2SO4], MgCl2, deoxynucleotide triphosphate mixture, and Taq DNA polymerase [Bioline (UK) Ltd.] and overlaid each reaction with 10 μL of mineral oil. Thus, each 13-μL PCR reaction contained the amount of the primers indicated in Table 1 and in addition, 1× PCR Buffer, 2mM MgCl2, 0.32 U of Taq DNA polymerase, 150 μM each deoxynucleotide triphosphate, and 0.08–0.15 μg of genomic DNA. Amplification was performed on a PTC-200 thermal cycler (GRI) using a high-stringency touchdown-PCR protocol with high annealing temperatures to ensure specificity of amplification. The PCR cycling condition were as follows: initial denaturation for 1 min at 96 °C; followed by 5 cycles of 20 s at 96 °C, 45 s at 70 °C, and 25 s at 72 °C; 21 cycles of 25 s at 96 °C, 50 s at 65 °C, and 30 s at 72 °C; 4 cycles of 30 s at 96 °C, 60 s at 55 °C, and 120 s at 72 °C. The PCR products were analyzed by electrophoresis on 2% Tris-borate-EDTA/ethidium bromide agarose gels with 10 μL of Orange G loading dye and visualized under ultraviolet illumination. For all PCR reactions (E2, E3, and E4), the presence of a 173-bp band indicated the presence of the specific ApoE haplotype. The control primer pair binds on two positions on chromosome 6, [on GI:20196696, MDL PRIMER-8 binds on sequences 53359–53341 and 89472–89454, whereas MDL PRIMER-9 binds on sequences 52575–52594 and 87875–87894]. Therefore, with the control primer pair, two products were expected of 785 and 1598 bp. Although the 785-bp control amplification product was always present when PCR amplification occurred, the 1598-bp product was often missing in those samples with poor-quality DNA or excessive ApoE haplotype-specific amplification. Nevertheless, a sample was considered negative for a particular ApoE haplotype when the haplotype-specific amplicon was absent and the 785-bp control amplicons was present. Absence of haplotype-specific and control amplicons in the same reaction was indicative of PCR amplification failure. Examples of ApoE haplotype-positive PCR reactions are shown in Fig. 1 . Amplification products of the six common ApoE genotypes by SSP-PCR. Primer mixture E2 contains MDL-Primers 1 and 3, which produce an ApoE-specific 173-bp product only if the nucleotides that encode for Cys112 and Cys158 are on the same chromosome. Primer mixture E3 contains MDL-Primers 1 and 2, which produce an ApoE-specific 173-bp product only if the nucleotides that encode Cys112 and Arg158 are on the same chromosome. Primer mixture E4 contains MDL-Primers 4 and 2, which produce an ApoE-specific 173-bp product only if the nucleotides that determine Arg112 and Arg158 are on the same chromosome. In all PCR reactions the control primer pairs produced the two larger PCR products as predicted, thus confirming PCR amplification in those PCR reactions that were devoid of allele-specific PCR products. The accuracy of this genotype determination method was demonstrated by analysis of a cohort of 50 DNA samples previously typed by the PCR-RFLP method and which included all ApoE haplotype combinations, examples of which are shown in Fig. 1 . For all samples, there was 100% concordance between the two methods. The SSP-PCR ApoE genotyping method performed equally well with DNA extracted by different methods, including Qiagen extraction and the high-salt method, and with variable amounts of DNA starting material. PCR amplification problems were observed when EDTA was included in the DNA resuspension/elution buffer. The methodology was easily adaptable to high-throughput genotyping by a combination of automatic DNA extraction using the QIAGEN MDX 8000 system, PCR plate technology, and 96-well-compatible agarose gel electrophoresis. With this system, the DNA of 96 samples could be extracted and the ApoE haplotype determined in a single run. This work utilized facilities made available through a new service and innovative grant from the Guy’s and St. Thomas’ Charitable Foundation for setting up the Pan-Pathology Molecular Diagnostic Laboratory.