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Technical Briefs |
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Applied Biosystems Div. of Perkin-Elmer, Foster City, CA;
a address for correspondence: Franz Volhard Clin., Wiltberg Str. 50, 13122 Berlin, Germany, fax 0049 30 9417 2206, e-mail fcluft{at}mdc-berlin.de
Apolipoprotein (apo) E is a protein component of lipoproteins,
50% of which resides in HDL, 10% in LDL, 20% in IDL, and 20% in
VLDL cholesterol fractions (1). Apo E binds to the LDL
receptor, also termed the B,E receptor, because the receptor accepts
both apo B and apo E. Apo E is also thought to bind to a specific
chylomicron remnant receptor by virtue of its structural determinants.
The heterogeneity in receptor binding of different varieties of apo E
is explained by the affinity of different apo E alleles to various
receptors. Apo E polymorphisms may be explained by three major alleles:
apo E
2, apo E3, and apo E4, which are found in 10%, 76%, and
13%, respectively, of the Caucasian population (2). The
polymorphisms are due to substitution of a cysteine for an arginine at
residue 112 or 158, or at both residues. The apo E2 variant has the
lowest affinity for the LDL receptor. There is an LDL concentration
gradient in both the healthy population and in those with coronary
heart disease. Individuals homozygous for apo E2 have the lowest
concentrations of LDL, and apo E4 homozygotes have the highest LDL
concentrations (3). The apo E4 allele has also been
associated with Alzheimer disease. However, the mechanisms of this
association are not yet clear (4). Thus, the interest in
apo E polymorphisms is high, both on the basis of epidemiological
research and for the purpose of clarifying individual lipid
disturbances or dementias. We have successfully applied the
oligonucleotide ligation assay (OLA) technique to screen for mutations
causing familial hypercholesterolemia (5). We have now
adapted this technique for the detection of apo E polymorphisms in
large numbers of samples.
PCR amplification of genomic DNA from peripheral blood was performed in a Perkin-Elmer 9600 Thermocycler. A 310-bp fragment of the apo E gene was amplified with the following primer sequences: 5'-GAG ACG CGG GCA CGG CTG TC-3'(upper primer) and 5'-GCA CGC GGC CCT GTT CCA C-3'(lower primer). The PCR reactions were performed in a total volume of 20 µL containing 10 mmol/L Tris-HCl pH 8.3, 50 mmol/L KCl, 4.5 mmol/L MgCl2, 0.1 g/L gelatin, 50 g/L dimethyl sulfoxide, 200 µmol/L each dNTP, 0.1 µmol/L each primer, 5 ng of genomic DNA, and 0.6 U of AmpliTaq Gold DNA polymerase (Applied Biosystems Division of Perkin-Elmer). Amplification conditions consisted of an initial 10-min denaturation at 95 °C followed by 40 cycles of 94 °C for 30 s, 68 °C for 30 s, 72 °C for 1 min, and a final extension of 5 min at 72 °C. An 8-µL aliquot of PCR product was used for OLA analysis.
The ethylene oxide mobility modifier chemistry was chosen because of several desirable properties (6). The probes are characteristically hydrophilic as required for a solution-phase enzymatic reaction. In addition, they are nonnucleotidic and partially negatively charged, to minimize potential nonspecific interactions with the oligonucleotide portion of the probe or with the target DNA. As a consequence, the mobility-modifying tails affect the Tm of the probes very little. The synthesis of this type of probe can be accomplished efficiently by using standard automated DNA synthesis chemistry (7)(8). We have found it best to use pentaethyleneoxide (PEO) modifiers, because the corresponding phosphoramidite monomers can be synthesized more effectively than the hexaethyleneoxide monomers (David G. Sherman, Applied Biosystems, personal communication).
OLA reactions for two polymorphic sites, located in the apo E gene,
were multiplexed in one tube. Three synthetic oligonucleotide probes
were used for the analysis of each polymorphic site, two allelic probes
with 5'-PEO tails of different length, and one common, 3'-fluorescently
labeled reporter probe. The sequences of the probes and the numbers of
mobility modifiers are given in Table 1
. OLA reactions were carried out in a 20-µL reaction volume
containing 20 mmol/L Tris-HCl pH 7.6, 25 mmol/L potassium acetate, 10
mmol/L magnesium acetate, 10 mmol/L dithiothreitol, 1 mmol/L NAD, 1
mL/L Triton X-100, 1–20 nmol/L each oligonucleotide probe, 8 µL of
PCR product, and 4 U of thermostable Thermus aquaticus
ligase (New England Biolabs). Linear amplification of product was
achieved by 20 cycles of 94 °C for 30 s and 45 °C for 3 min,
followed by heating at 99 °C for 10 min in a Perkin-Elmer 9600
Thermocycler.
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A 2-µL aliquot of each multiplex OLA product was mixed with 2.5 µL of deionized formamide, 0.5 µL of dextran blue loading buffer, and 0.5 µL of Genescan-500 Tamra size marker. The mixture was denatured at 95 °C for 3 min and then rapidly cooled on ice before loading the gel. OLA products were electrophoresed for 2.5 h at 2500 V on an automated Model 373A fluorescence-scanning DNA sequencer (Applied Biosystems Division of Perkin-Elmer) with an 8% acrylamide, 19:1 acrylamide:bisacrylamide denaturing gel containing 8.3 mol/L urea, 89 mmol/L Tris, 89 mmol/L boric acid, and 2 mmol/L EDTA. The gel thickness was 0.4 mm and the well-to-read length was 24 cm. The resulting gel data were analyzed for peak color and fragment size by using the Genescan 672 fragment analysis software and the Genotyper software (Applied Biosystems Division of Perkin-Elmer).
Figure 1
shows OLA results from six probands with the six possible apo E
genotypes. After PCR amplification of the apo E gene fragment that
contains the two polymorphic sites of interest, competitive OLA is run
at both loci. For each polymorphic site, two allele-specific
oligonucleotide probes and a third, common probe are hybridized to one
strand of the PCR product such that the 3' ends of the allele-specific
probes are immediately adjacent to the 5' end of the common probe. This
sets up a competitive hybridization–ligation process between the two
allelic probes and the common probe. The thermostable DNA ligase then
discriminates between single-base mismatches at the junction site
(9), thereby producing allele-specific ligation products.
Since the common probe is labeled with a fluorescent dye and the
allele-specific probes are linked to different-length PEO
mobility-modifying tails, each ligation product can be identified by
its defined electrophoretic mobility and fluorescent color on a
four-color fluorescence-scanning sequencer. The mobility modifiers
allow the mobility of each ligation product to be arbitrarily defined,
regardless of oligonucleotide length or sequence. They are oligomeric,
making them suitable for assembly onto the probes during automated
oligonucleotide synthesis. This approach to multiplex analysis is
termed "sequence-coded separation" to highlight both the decoupling
of mobility from the physical size of the ligated probe and the
deliberately designed linkage between mobility and target sequence
(6). The sequences of the oligonucleotides we used and the
ligation product sizes are given in Table 1
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After gel electrophoresis is completed, the Genescan and Genotyper software are used to analyze the gel data. Lanes are defined and fragments are sized. The probands had been genotyped with conventional methods earlier. The results were the same in every instance, assuring the desired sensitivity and specificity of the method. In addition to a global quality check, the entire analysis, namely sizing, allele calling, and reporting, is automated. The results are automatically compiled and transferred by computer to our computerized clinical data base. This approach allows correlations to be drawn with the clinical findings, which allows the clinician to make reasoned judgments regarding clinical care.
Our data demonstrate the utility and power of the OLA technology in identifying apo E polymorphisms. PCR-based procedures have been used by others to identify the underlying mutations in apo E (3)(4). The OLA approach facilitates our diagnostic capabilities above and beyond the earlier methodology. The new method is sensitive, specific, reproducible, fast, and flexible (5). The OLA technique will permit epidemiologists to determine the frequency and heterogeneity of apo E mutations in population studies.
The OLA assay can be done quickly because of the single tube and single gel lane format. Furthermore, the Genotyper software method makes the OLA objective and independent from interference by users. Thus, quality control can be easily determined, which simplifies the application of this method for clinical care.
We have adapted the OLA method to the point that blood sampling can be accomplished with a few drops of blood onto filter paper or even from a salivary sample (10)(11). Thus, the samples can be shipped through the mail inside a simple envelope. This feature makes the OLA particularly attractive for large-scale epidemiological investigations and studies in population genetics. Obviously, a strategy must be applied that takes into consideration informed consent and rights of privacy. We have developed such approaches (unpublished).
Interest in apo E polymorphisms has recently increased greatly because
of the association of apo E4 with Alzheimer disease. Heart
disease is increased in patients with Alzheimer disease and the
increased occurrence of both diseases has been linked to the presence
of the apo E4. The OLA method may move genetic testing for apo E
polymorphisms into the clinical arena. The test may not be appropriate
for the routine clinical laboratory; however, reference laboratories
will be in a position to provide this service for large numbers of
patients because of its automation. In our view, apo E polymorphisms
should not be routinely tested in individuals for clinical purposes.
Many patients develop Alzheimer disease who do not have the apo E4
allele, and most persons with apo E4 appear to escape Alzheimer
disease. Thus, at the present time, testing for apo E4 in routine
clinical diagnosis is not recommended and apo E4 should not be used
for predictive testing (12). However, there are ample
reasons to examine apo E polymorphisms for research purposes. If large
population samples, such as the Framingham cohort, are to be
tested, the OLA technology provides a highly specific and powerful
assay to get the job done efficiently.
Acknowledgments
We thank Eric Shulse for his support and encouragement of the project, and Tess Adriano and James Liang for their technical help with the probe synthesis. H.S., H.B., and A.A. are supported by the Deutsche Forschungsgemeinschaft. F.C.L. is supported by a grant-in-aid from the the Bundesministerium für Bildung und Forschung.
Footnotes
Franz Volhard Clin. at the Max Delbrück Center for Molec. Med., Virch. Klin., Humboldt Univ. of Berlin, Berlin, Germany
References
The following articles in journals at HighWire Press have cited this article:
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  E. E. Niederkofler, K. A. Tubbs, U. A. Kiernan, D. Nedelkov, and R. W. Nelson Novel mass spectrometric immunoassays for the rapid structural characterization of plasma apolipoproteins J. Lipid Res., March 1, 2003; 44(3): 630 - 639. [Abstract] [Full Text] [PDF]
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 N. Jacobsen, J. Bentzen, M. Meldgaard, M. H. Jakobsen, M. Fenger, S. Kauppinen, and J. Skouv LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E Nucleic Acids Res., October 1, 2002; 30(19): e100 - e100. [Abstract] [Full Text] [PDF]
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 M. Nauck, M. M. Hoffmann, H. Wieland, and W. Marz Evaluation of the Apo E Genotyping Kit on the LightCycler, Clin. Chem., May 1, 2000; 46(5): 722 - 724. [Full Text] [PDF]
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E.-L. Romppanen and I. Mononen PCR-Oligonucleotide Ligation Assay from Dried Blood Spots Clin. Chem., November 1, 1999; 45(11): 2022 - 2025. [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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