Clinical Chemistry 43: 1321-1324, 1997;
(Clinical Chemistry. 1997;43:1321-1324.)
© 1997 American Association for Clinical Chemistry, Inc.
Apolipoprotein E genotyping by capillary electrophoretic analysis of restriction fragments
Gianluca De Bellis1,a,
Giuliana Salani1,
Silvia Panigone1,
Ferruccio Betti1,
Luigia Invernizzi1 and
Massimo Luzzana2
1
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche Avanzate, Via Fratelli Cervi 93, 20090 Segrate, Italy.
2
Università degli Studi di Milano, Dipartimento di
Scienze e Tecnologie Biomediche, Via Fratelli Cervi 93, 20090 Segrate,
Italy.
a Author for correspondence. Fax 39-2-26422770; e-mail debellis{at}itba.mi.cnr.it
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Abstract
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We present the genotyping of apolipoprotein (apo) E by means of
restriction fragment analysis of amplified genomic DNA by
high-performance capillary electrophoresis and a replaceable
non-gel-sieving matrix. This procedure streamlines the genotyping of
apo E in large-scale population studies because of the automation and
speed of capillary electrophoresis.
Key Words: indexing terms: genotype determination • Alzheimer disease • high-performance capillary electrophoresis
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Introduction
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Apolipoprotein (apo) E is an important constituent of several
plasma lipoproteins, mainly VLDL and HDL and
chylomycrons.1
It is involved in the
redistribution of lipids in the liver and is implicated in growth and
repair of injured neurons in the nervous system (1). Apo E
has been associated with the risk of developing cardiovascular diseases
and in familial type III hyperlipoproteinemia (2). More
recently a strong association between apo E and Alzheimer disease has
been demonstrated (3). Human apo E exists in three main
isoforms (E2, E3, and E4) related to two polymorphic sites at codons
112 and 158 of the gene located on chromosome 19 (Table 1
). These isoforms arise from three alleles (
2, 3, and 4
respectively) combined in six different genotypes. The E4 isoform has
been associated with Alzheimer disease as a major risk factor. In
particular in late-onset Alzheimer subjects with known familial
occurrence, the 4 allele frequency is much higher than in
age-matched individuals, whereas 2 is much lower (4).
Furthermore the age of onset of the disease is related to the 4
allele dose (3). Over 40 independent studies discussing
apo E/Alzheimer association in different populations have been
published so far with comparable results (5).
View this table:
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Table 1. Correspondence among apo E protein isoforms, amino acid and
codon composition at polymorphic sites, and
genotypes.
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Alzheimer disease has a severe impact in the elderly population
worldwide. Large studies to follow the elderly population with respect
to this pathology are currently under way, as are clinical trials to
test drugs with benefical effects on affected individuals. This makes
the typing of apo E isoforms very important in Alzheimer studies. An
interesting recent review focused on apo E relevance in laboratory
medicine and reported the complete range of analytical techniques
devoted to the apo E polymorphism determination (6).
Phenotyping is usually performed by means of isoelectrofocusing
techniques, but these present several drawbacks in their application to
this specific analytical problem. Apo E genotyping has been developed
to avoid such problems. Several recent papers dealt with the analysis
of the apo E genotype by means of PCR amplification from genomic DNA
(6). Most of the cited studies involved restriction
fragment analysis of the amplified region encompassing codons 112 and
158 of the apo E gene (7)(8). This procedure
has been widely adopted, although it has some drawbacks particularly
related to complex electrophoretic pattern due to partial enzymatic
digestion and to the requirement of large quantities of amplified DNA
(9). Modified procedures have been proposed to overcome
such problems (9)(10).
Here we present the identification of the apo E genotype by means of
capillary electrophoresis analysis. A relatively viscous sieving media
is required to achieve a good separation of the relatively small DNA
fragments (48–91 bp) to be analyzed. High-performance capillary
electrophoresis (HPCE) with cross-linked polyacrylamide gel offers the
best performance in terms of resolution, and recently two groups
(11)(12) proposed such a technique for apo E
genotyping. However, such a sieving matrix cannot be replaced. Several
non-gel-sieving media, prepared from polymers, have been proposed to
separate DNA molecules. These have a key feature in that they are
replaceable after each separation, thus performing very reproducibly
from run to run. By using methyl cellulose as a replaceable sieving
matrix, the digestion fragments are sized in a very short time (10 min)
with a commercial automated apparatus for capillary electrophoresis
with UV on-line detection. We present results demonstrating the speed,
sensitivity, and reliability of this analytical procedure.
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Materials and Methods
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The preparation of the sample for the determination of the apo E
genotype followed a well-estabilished procedure
(7)(8) with minor modifications. Samples (178)
were processed at different times. DNA extraction from whole blood was
performed with a commercial kit based on chromatographic columns. This
material was amplified, giving the expected DNA fragment. The amplified
samples were checked for purity and quantity on a standard agarose gel
(10 g/L) and digested with CfoI, an isoschizomer of
HhaI.
dna extraction and apo e amplification
DNA was extracted and purified from 200 µL of whole blood, drawn
from consenting individuals, with the blood DNA extraction kit (Qiagen,
Chatsworth, CA). DNA was collected in 200 µL of water (25–150 ng/L)
and stored at 4 °C. Oligonucleotides P1 and P2 (7) used
for the amplification of the polymorphic region were purchased from
Genset (Paris, France), diluted to 20 µmol/L, and stored at
-20 °C. The DNA amplification was performed in a thermal cycler
(Perkin-Elmer, Norwalk, CT) in a total volume of 100 µL. The
amplification mixture contained Taq buffer diluted 1:10 as suggested by
the manufacturer, deoxynucleotides (200 µmol/L each), 40 pmol of each
primer, and 8 µL of purified genomic DNA. The mixture was overlaid
with mineral oil and heated at 95 °C for 10 min. Taq polymerase (2.5
U; Finnzyme, Espoo, Finland) was then added and the samples were
subjected to five cycles as follows: 95 °C for 1 min and 72 °C
for 3 min and then to 30 cycles as follows: 95 °C for 1 min,
60 °C for 1 min, and 72 °C for 1 min. Samples were then incubated
at 72 °C for 10 min and stored at 4 °C.
dna digestion and purification
After the amplification, 40 U of CfoI (Boehringer
Mannheim, Mannheim, Germany) were added and the samples were digested
overnight at 37 °C. Before digestion, the sample can be purified by
means of Microspin columns (S200; Pharmacia, Uppsala, Sweden) to remove
nucleotides, primers, and PCR buffer. The digested sample was then
isopropanol-precipitated or ultrafiltered (we recommend this procedure)
by means of MC 5000 columns (Millipore, Bedford, MA), washing two times
with 400 µL of water. The resulting solution was adjusted to 20 µL
with water.
capillary electrophoresis analysis
Analysis was performed on a BioFocus 3000 capillary
electrophoresis system (Bio-Rad, Hercules, CA) by means of a standard
cartridge containing a 50 cm x 50 µm coated capillary tube
thermostated at 20 °C. The capillary was washed with TBE 1x (89
mmol/L Tris base, 89 mmol/L boric acid, and 1 mmol/L EDTA disodium
salt) and filled with a 12 g/L solution of Methocel (Methylcellulose
high density; Fluka, Buchs, Switzerland) in TBE 1x. This solution was
prepared as follows: 1 g of Methocel was slowly added to water (50
mL) at 90 °C. The solution was allowed to cool at room temperature
under constant stirring for 30 min and then cooled at 4 °C in a
refrigerator, continuing the stirring. After 2 h it was
centrifuged, filtered through a 0.45-µm filter, and stored at
4 °C. The solution was mixed with TBE 10x, and adjusted with water
to a final concentration of 12 g/L in TBE 1x.
Samples were electroinjected at 10 kV for 5 s. Restriction
fragments were separated during a 10-min run at 15 kV and detected at
260 nm.
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Results
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The digestion of the amplified region encompassing the two
polymorphic sites of interest generates a number of fragments. Fig. 1
A shows the restriction map for this region with respect to the
sequence of the three most common alleles. The expected electrophoretic
pattern for the six resulting genotypes is presented in Fig. 1B
. Only
the DNA fragments ranging from 48 to 91 bp are diagnostic for the
genotyping of this locus.
After some trials we selected methylcellulose (high viscosity) as the
replaceable sieving media to be used in capillary electrophoresis
analysis of these relatively short DNA fragments. We optimized the
separation variables by means of a DNA size marker prepared by
CfoI digestion of a commercial preparation of pUC18
(fragments ranging from 393 to 21 bp). This DNA ladder was analyzed by
changing several variables (applied voltage, temperature, polymer
concentration, sample injection conditions, capillary tube length,
diameter, and coating). A standard 50 cm x 50 µm coated
capillary column (Bio-Rad) was found to be appropriate for high
resolution and speed of analysis. Alternatively, the analysis was
performed on a 50 cm x 50 µm DB-1 coated capillary column (J&W
Scientific, Folsom, CA). As shown in Fig. 2
, the diagnostic 48-, 63-, 72-, 82-, and 91-bp bands are well
resolved in <10 min. All six common genotypes can be easily
discriminated. The run-to-run reproducibility was tested by injecting
the same sample 30 times, refilling the capillary tube each time. The
CV for the retention time is 2.2%. CV for peak area is 21.7%. With an
external calibrator (bromophenol blue), the CV for normalized peak area
is 5.6%. The filling of the capillary takes 5 min (50 cm x 50
µm column) and therefore the reuse of the sieving matrix would be
desirable to speed up the analysis. We tested this possibility by
injecting the same sample several times and performing the analysis as
explained without sieving matrix reneval. The first six injections gave
a CV for the retention time within that reported, indicating that
multiple use of the matrix is feasible.
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Discussion
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The reliability of apo E genotyping by restriction digestion and
polyacrylamide gel electrophoresis (PAGE) has been recently discussed.
Some important drawbacks can affect the analysis. Because of partial
digestion, "ghost" bands appear in PAGE, biasing the correct
genotyping. Bands having different intensities have been indicated as a
possible source of problems during ethidium bromide/PAGE analysis
(9). In addition, relevant quantities of amplified DNA
should be loaded on the gel because of the intrinsic low sensitivity of
ethidium bromide staining with small DNA fragments. Appel et al.
(9) recently proposed an alternative procedure requiring
radioactive labeling. In an attempt to overcome such problems we used
HPCE for the analysis of the restriction fragments generated by
HhaI (CfoI) digestion. This technique was
recently proposed by Baba et al. (11) and Schlenck et al.
(12) for apo E genotyping. Both used a cross-linked
polyacrylamide gel as separation medium, thus gaining very high
resolution (12). UV- (11) or laser-induced
fluorescence (12) was used as detection system.
The resolution we obtained with methylcellulose was lower than that
gained by polyacrylamide (12), although it is sufficient
to discriminate all six common apo E genotypes. However, such a sieving
matrix yielded very reproducible results because of the possibility of
its renewal after runs, whereas polyacrylamide capillary columns
decrease their performance during their lifetime.
We used UV detection, gaining good sensitivity. PCR usually gave 400 ng
of amplified DNA that was precipitated, digested, desalted, and
electroinjected several (10–30) times, yielding reproducible
electropherograms similar to those shown in Fig. 2
. This was achieved
without the need for an expensive laser apparatus for fluorescence
detection, although this grants a much higher sensitivity
(12). We observed a large difference comparing the signal
intensity we obtained with that shown in ref. 11. This is
probably due to the accurate desalting of digested samples performed
before electroinjection. This allows an on-line concentration,
particularly for smaller fragments.
HPCE, giving a quantitative estimate of each band by UV on-line
detection, helps in preventing genotyping errors due to partial
digestion. In fact, peak integration allows the comparison of the
relative intensities of the bands on a numerical basis. Although the
peak intensity ratio can vary because of the inherent variability of
electrokinetic injection, the presence of "ghost" bands, like that
in Fig. 2F
(the shoulder close to the 63-bp fragment), can be easily
detected and correctly classified. Samples that are difficult to
discriminate, because of partial digestion, can be directly redigested
and analyzed because of the extremely small sample quantities required
by the capillary electrophoresis analysis. Sample loading by
electroinjection has proven to be very efficient, although it requires
a preliminary optimization with respect to the actual concentration of
the sample. The precipitation performed after digestion yields very
concentrated samples that must be injected for a very short time to
avoid overload and loss of resolution. Despite this fact, it is very
simple to determine the genotype in very faint samples by means of
longer injection time, thus overcoming the sensitivity problem. The
injection of the same sample 30 times demonstrates the sensitivity of
this approach and its reliability (low CV for retention time). The
speed of the analysis can be further increased by shortening the
capillary tube. In a 20-cm x 50-µm tube, acceptable separation
was gained in <3 min (data not shown). Preparation of the sieving
matrix has proven to be critical to the final result. The reported
procedure must be followed carefully as explained to yield consistent
results. The results achieved with this method in our laboratory,
during a large-scale apo E genotype study, are extremely positive. On
some occasions, in partially digested samples, genotyping by PAGE and
by HPCE were in disagreement, but after further digestion, the
genotyping by HPCE was confirmed.
In conclusion, this procedure could be of interest to
clinical laboratories involved in large-scale apo E genotype analysis
(6), thanks to the high degree of automation attainable
with HPCE combined with the speed, sensitivity, and reliability of the
proposed analytical protocol.
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Acknowledgments
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We gratefully acknowledge Telethon (grant E213) for
partial financial support.
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Footnotes
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1 Nonstandard abbreviations: apo, apolipoprotein; HPCE,
high-performance capillary electrophoresis; TBE, Tris–boric acid–EDTA
buffer; and PAGE, polyacrylamide gel electrophoresis. 
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Abstract
Introduction
