Discrepancy in Genotyping of Apolipoprotein E between Allele-Specific PCR and Fluorescence Resonance Energy Transfer or Sequencing

Chang-Hun Park; Seung-Tae Lee; Chang-Seok Ki; Jong-Won Kim

doi:10.3343/kjlm.2010.30.3.325

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Park, Lee, Ki, and Kim: Discrepancy in Genotyping of Apolipoprotein E between Allele-Specific PCR and Fluorescence Resonance Energy Transfer or Sequencing

Brief Communication

Korean J Leg Med 2010;30(3):325-328.

Published online: 14 January 2010

DOI: https://doi.org/10.3343/kjlm.2010.30.3.325

Discrepancy in Genotyping of Apolipoprotein E between Allele-Specific PCR and Fluorescence Resonance Energy Transfer or Sequencing

Chang-Hun Park, M.D., Seung-Tae Lee, M.D., Chang-Seok Ki, M.D., Jong-Won Kim, M.D.

Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Corresponding author: Chang-Seok Ki, M.D. Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Korea Tel: +82-2-3410-2709, Fax: +82-2-3410-2719 E-mail: changski@skku.edu

^∗ Results of sequencing analysis of specific primers that target a 526C nucleotide are shown to the right in cases 1 and 2.

Revised 8 January 201012 April 2010 Accepted 21 April 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The human apolipoprotein E (APOE) gene contains several single-nucleotide polymorphisms (SNPs) that are distributed across the gene. The genotype of the APOE gene has important implications as a risk factor for various diseases. We observed 2 cases in which the results of allele-specific PCR (AS-PCR) of the APOE gene were not consistent with those of fluorescence resonance energy transfer (FRET) or sequencing analysis. In these cases, genotyping by AS-PCR showed that patients were ε2 homozygotes, while sequencing analysis and FRET showed that they were ε2/ε3 heterozygotes. Herein, we describe the causes of the errors in genotyping and describe the significance of these errors.

Keywords: APOE, Allele-specific PCR, Fluorescence resonance energy transfer, Genotyping, Sequencing

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Fig. 1.

A flowchart summarizing the analytic courses and results for the 673 cases.

Fig. 2.

Agarose gel electrophoresis of AS-PCR products. Case 1 and case 2 were ε2/ε2 homozygous. Abbreviation: M, APOE marker.

Fig. 3.

Detection of mutations in the APOE gene by PCR-direct sequencing analyses. Case 1 and case 2 showed 388TT (left), 526TC (middle), and 2 point hemizygous mutations (784A∗ and 787A∗) (right).

Fig. 4.

PCR bands of each allele (ε2, ε3, ε4, and ε7) and their interpretations in AS-PCR. The ε7 alleles of genotypes ε3/ε7 and ε4/ε7 were interpreted as ε3, and the ε7 allele of genotype ε2/ε7 was interpreted as ε2 (dotted box).

Table 1.

Six different genotypes resulting from 3 polymorphic alleles (ε2, ε3, and ε4) of the APOE gene

Allele∗	Nucleotide number
Allele∗	388^†	526^†
ε2/ε2	T/T	T/T
ε2/ε3	T/T	T/C
ε2/ε4	T/C	T/C
ε3/ε3	T/T	C/C
ε3/ε4	T/C	C/C
ε4/ε4	C/C	C/C

^∗ ε2 (388T, 526T), ε3 (388T, 526C), ε4 (388C, 526C):

^† A of the ATG initiation codon was numbered+1 based on GenBank accession number NM_000041.

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