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Technical Briefs |
Inst. of Thrombosis and Hemostasis, Sheba Med. Center, Tel Hashomer, Israel
a author for
correspondence: fax 972-3-5351568,
Apolipoprotein E (apo E) is a protein that plays an essential role in lipid metabolism and distribution (1). The apo E gene is polymorphic, and its three alleles code for isoforms E2, E3, and E4, which differ by single-amino-acid substitutions (2). The apo E3 allele is the predominant isoform in all populations studied. The apo E4 allele is associated with increased total serum cholesterol and greater odds for coronary heart disease (3); it also constitutes a major risk factor for Alzheimer disease (4). The apo E2 allele seems to have a protective effect against Alzheimer disease and is associated with longevity (5). Consequently, interest in examining individual patients and study groups for the apo E isoforms is growing. In this communication we describe a simple procedure that facilitates the genotyping of the apo E polymorphisms.
In the common apo E3 polymorphism, TGC encodes for Cys112, and CGC encodes for Arg158. In the apo E2 another TGC codon results in Cys158, whereas in the apo E4 a different CGC codon gives rise to Arg112. The three apo E alleles determine six genotypes, i.e., three homozygotes designated E4/E4, E3/E3, and E2/E2 and three heterozygotes designated E3/E4, E2/E3, and E2/E4.
Early methods for detection of apo E isoforms were based on
protein isoelectrofocusing (6). After the identification
of the apo E gene (7) molecular methods based on PCR
amplification and HhaI digestion were introduced
(8)(9) and later somewhat improved
(10)(11). However, all PCR-based assays are
difficult to interpret because the HhaI enzyme yields
several small fragments, not all of which are specific for the apo E
genotypes. Moreover, incomplete digestion by HhaI can yield
ambiguous results. In this study we used two new restriction enzymes,
i.e., AflIII and HaeII, that recognize the
allele-specific nucleotide substitutions at codons 112 and 158,
respectively, and do not recognize additional sites. Fig. 1
A illustrates schematically a
loss of an AflIII restriction site that is characteristic
for the apo E4 allele and a loss of an HaeII restriction
site that is unique for the apo E2 allele (see asterisks).
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DNA was purified from leukocytes by the salting-out method as described (12). Genomic DNA was amplified by PCR with the primers F5'-TCCAAGGAGCTGCAGGCGGCGCA and R5'-GCCCCGGCCTGGTACACTGCCA to yield a 218-bp DNA fragment that spans both apo E polymorphic sites. In the PCR, 100–200 ng of DNA was added to 25 µL of reaction mixture containing 75 mmol/L Tris-HCl, pH 9.0, 20 mmol/L ammonium sulfate, 0.1 mL/L Tween, 1.5 mmol/L MgCl2, 500 nmol/L of each primer, 0.2 mmol/L dNTPs, 100 mL/L dimethyl sulfoxide, and 0.6 units of Taq polymerase (Advance Biotechnology).
The PCR reactions were subjected to 40 cycles in a thermal cycler (MJ Research) with 30 s of denaturing at 94 °C, 30 s of annealing at 55 °C, and 90 s of extension at 70 °C. Amplified DNA (15 µL) was digested simultaneously with 2.5 units of AflIII and 5 units of HaeII (New England Biologicals) for 24 h at 37 °C, analyzed on 4% agarose gel (metaPhor, FMC), and visualized by ethidium bromide staining.
As expected, simultaneous digestion of the 218-bp amplified
product yielded on 4% agarose gel electrophoresis 145-bp, 168-bp, and
195-bp fragments that were specific for apo E3, E2, and E4,
respectively (Fig. 1
). All six possible genotypes for apo E, i.e.,
E2/E4, E4/E4, E3/E4, E3/E3, E2/E3, and E2/E2, were clearly discernible
(lanes 2–7, respectively). In the E2/E4 genotype (Fig. 1B
, lanes 2 and
8) a residual uncut 218-bp fragment was present. To characterize the
nature of this uncut fragment, the following experiments were carried
out. When the PCR product of the genotype E2/E2 was mixed with the PCR
product of the genotype E4/E4 and subjected to simultaneous digestion
with AflIII and HaeII, only the bands
corresponding to alleles E2 (168 bp) and E4 (195 bp) were observed
(Fig. 1B
, lane 9). In contrast, when the same mixture of E2/E2 plus
E4/E4 was allowed to denature (95 °C for 5 min) and anneal at
55 °C before digestion, the uncut 218-bp band was observed (Fig. 1B
, lane 10). These findings were consistent with a heteroduplex formation
between DNA strands carrying the E2 and the E4 sequences with its
anticipated resistance to the enzyme digestion.
In conclusion, simultaneous digestion of an amplified segment of the apo E gene by AflIII and HaeII enzymes clearly determines all apo E genotypes. The assay is easy to perform and can be used for analysis of numerous samples within a short time.
References
The following articles in journals at HighWire Press have cited this article:
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  S. M. Laws, R. Perneczky, A. Drzezga, J. Diehl-Schmid, B. Ibach, J. Bauml, T. Eisele, H. Forstl, A. Kurz, and M. Riemenschneider Association of the Tau Haplotype H2 With Age at Onset and Functional Alterations of Glucose Utilization in Frontotemporal Dementia Am J Psychiatry, October 1, 2007; 164(10): 1577 - 1584. [Abstract] [Full Text] [PDF]
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 J.-M. Kim, R. Stewart, S.-W. Kim, S.-J. Yang, I.-S. Shin, and J.-S. Yoon A Prospective Study of Changes in Subjective Memory Complaints and Onset of Dementia in South Korea. Am J Geriatr Psychiatry, November 1, 2006; 14(11): 949 - 956. [Abstract] [Full Text] [PDF]
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 M. Riemenschneider, L. Konta, P. Friedrich, S. Schwarz, K. Taddei, F. Neff, A. Padovani, H. Kolsch, S. M. Laws, N. Klopp, et al. A functional polymorphism within plasminogen activator urokinase (PLAU) is associated with Alzheimer's disease Hum. Mol. Genet., August 15, 2006; 15(16): 2446 - 2456. [Abstract] [Full Text] [PDF]
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 N. Ghebranious, L. Ivacic, J. Mallum, and C. Dokken Detection of ApoE E2, E3 and E4 alleles using MALDI-TOF mass spectrometry and the homogeneous mass-extend technology Nucleic Acids Res., October 4, 2005; 33(17): e149 - e149. [Abstract] [Full Text] [PDF]
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 M Pinholt, J L Frederiksen, P S Andersen, and M Christiansen Apo E in multiple sclerosis and optic neuritis: the Apo E-o4 allele is associated with progression of multiple sclerosis Multiple Sclerosis, October 1, 2005; 11(5): 511 - 515. [Abstract] [PDF]
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 A. Drzezga, T. Grimmer, M. Riemenschneider, N. Lautenschlager, H. Siebner, P. Alexopoulus, S. Minoshima, M. Schwaiger, and A. Kurz Prediction of Individual Clinical Outcome in MCI by Means of Genetic Assessment and 18F-FDG PET J. Nucl. Med., October 1, 2005; 46(10): 1625 - 1632. [Abstract] [Full Text] [PDF]
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 M. L. Slattery, C. Sweeney, M. Murtaugh, K. N. Ma, J. D. Potter, T. R. Levin, W. Samowitz, and R. Wolff Associations between apoE genotype and colon and rectal cancer Carcinogenesis, August 1, 2005; 26(8): 1422 - 1429. [Abstract] [Full Text] [PDF]
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 A. Drzezga, M. Riemenschneider, B. Strassner, T. Grimmer, M. Peller, A. Knoll, S. Wagenpfeil, S. Minoshima, M. Schwaiger, and A. Kurz Cerebral glucose metabolism in patients with AD and different APOE genotypes Neurology, January 11, 2005; 64(1): 102 - 107. [Abstract] [Full Text] [PDF]
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 W. Marz, H. Scharnagl, M. M. Hoffmann, B. O. Boehm, and B. R. Winkelmann The apolipoprotein E polymorphism is associated with circulating C-reactive protein (the Ludwigshafen risk and cardiovascular health study) Eur. Heart J., December 1, 2004; 25(23): 2109 - 2119. [Abstract] [Full Text] [PDF]
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H. Wang, J. Li, H. Liu, Q. Liu, Q. Mei, Y. Wang, J. Zhu, N. He, and Z. Lu Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film Nucleic Acids Res., June 15, 2002; 30(12): e61 - e61. [Abstract] [Full Text] [PDF]
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M. Riemenschneider, S. Wagenpfeil, J. Diehl, N. Lautenschlager, T. Theml, B. Heldmann, A. Drzezga, T. Jahn, H. Forstl, and A. Kurz Tau and A{beta}42 protein in CSF of patients with frontotemporal degeneration Neurology, June 11, 2002; 58(11): 1622 - 1628. [Abstract] [Full Text] [PDF]
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 M Riemenschneider, J Diehl, U Muller, H Forstl, and A Kurz Apolipoprotein E polymorphism in German patients with frontotemporal degeneration J. Neurol. Neurosurg. Psychiatry, May 1, 2002; 72(5): 639 - 641. [Abstract] [Full Text] [PDF]
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 A. Inbal, D. Freimark, B. Modan, A. Chetrit, S. Matetzky, N. Rosenberg, R. Dardik, Z. Baron, and U. Seligsohn Synergistic Effects of Prothrombotic Polymorphisms and Atherogenic Factors on the Risk of Myocardial Infarction in Young Males Blood, April 1, 1999; 93(7): 2186 - 2190. [Abstract] [Full Text] [PDF]
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 G. G. Donohoe, A. Salomaki, T. Lehtimaki, K. Pulkki, and V. Kairisto Rapid Identification of Apolipoprotein E Genotypes by Multiplex Amplification Refractory Mutation System PCR and Capillary Gel Electrophoresis Clin. Chem., January 1, 1999; 45(1): 143 - 146. [Full Text] [PDF]
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 O. Shpilberg, I. Rabi, K. Schiller, R. Walden, D. Harats, K.S. Tyrrell, B. Coller, and U. Seligsohn Patients With Glanzmann Thrombasthenia Lacking Platelet Glycoprotein {alpha}IIb{beta}3 (GPIIb/IIIa) and {alpha}v{beta}3 Receptors Are Not Protected From Atherosclerosis Circulation, March 5, 2002; 105(9): 1044 - 1048. [Abstract] [Full Text] [PDF]
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X174 was used as a
standard.