Clinical Chemistry 45: 771-776, 1999;
(Clinical Chemistry. 1999;45:771-776.)
© 1999 American Association for Clinical Chemistry, Inc.
Optimization of Apolipoprotein(a) Genotyping with Pulsed Field Gel Electrophoresis
Pieter H. Griffioen1,
Louwerens Zwang1,
Ron H.N. van Schaik1,
Henk Engel2,
Jan Lindemans1,a and
Christa M. Cobbaert3
1
University Hospital Rotterdam Dijkzigt, Department of Clinical Chemistry, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
2
St. Sophia Hospital, Department of Clinical Chemistry,
P.O. Box 10400, 8000 GK Zwolle, The Netherlands.
3
Hospital De Baronie, Department of Clinical Chemistry,
P.O. Box 90157, 4800 RL Breda, The Netherlands.
a Author for correspondence. Fax 31-10-4367894; e-mail lindemans{at}ckcl.azr.nl
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Abstract
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Background: Increased lipoprotein(a) is a risk factor for
atherosclerosis, and its concentration in serum is inversely correlated
with the size of the apoliprotein(a) [apo(a)] component. The size of
the apo(a) gene is determined mainly by the Kringle IV size
polymorphism. We have optimized and characterized pulsed field gel
electrophoresis (PFGE) for apo(a) genotyping.
Methods: Established PFGE protocols were adjusted. The changes
included the following: (a) increased DNA yields by the
use of all leukocytes for isolation from either 3 mL of fresh EDTA
whole blood or 250 µL of frozen buffy coats; (b)
increased efficiency of Kpn1 digestion by the inclusion
of a digestion buffer wash; (c) reduction of assay time
by the use of capillary blotting; (d) increased
sensitivity by the use of four digoxigenin-labeled apo(a) probes; and
(e) identification using a single film by the inclusion
of a digoxigenin-labeled lambda marker probe in addition to apo(a)
probes in the hybridization mix.
Results: In older Caucasians, 93% (buffy coats, n=468) were
heterozygous for apo(a) gene size. An inverse correlation between serum
lipoprotein(a) and the sum of Kringle IV alleles was found
(y = -23x + 1553;
r = -0.442; n = 468). Gel-to-gel variation
was minimal (3%). Imprecision (SD) was one Kringle IV repeat (control
sample containing eight fragments of 72–233 kb; n=34 electrophoretic
runs).
Conclusions: The practicality and sensitivity of the apo(a)
genotyping technique by PFGE were improved, and accuracy and
reproducibility were preserved. The optimized procedure is promising
for apo(a) genotyping on frozen buffy coats from large epidemiological
studies.© 1999 American Association for Clinical
Chemistry
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Introduction
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Increased lipoprotein(a)
[Lp(a)]1
has been established as one of the major risk factors for
atherosclerosis (1)(2). Lp(a) serum
concentrations were shown to be inversely correlated with the size of
the apolipoprotein (a) [apo(a)] component(3)(4), which is determined mainly by the number
of Kringle IV repeats (5). apo(a) gene size
polymorphisms can be detected with pulsed field gel electrophoresis
(PFGE) (5). In currently described protocols, DNA is
isolated from 10 mL (6) or 16 mL (5) of fresh
EDTA blood, and the apo(a) genotype is determined using radioactive(5) or digoxigenin-labeled (6) probes. In ongoing
clinical studies of the determinants of coronary artery disease, we
wanted to use either tiny EDTA blood samples or frozen buffy coats as
the starting material for the determination of apo(a) gene size.
Established procedures were adjusted and subsequently tested for
accuracy and reproducibility. Modifications were made with respect to
the amount and type of starting material, the efficiency of digestion,
the sensitivity of chemiluminescent detection, and the practicability.
We verified the accuracy of the modified method using plugs containing
DNA for which the apo(a) genotype had been established in advance by
Trommsdorff et al. (6). We tested the reproducibility of the
modified method by running a self-manufactured control sample on each
gel throughout the study. We concluded that the proposed modifications
enhance the sensitivity, reproducibility, and practicality of the
technique for apo(a) genotyping.
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Materials and Methods
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dna specimens and patient sera
Fresh EDTA blood and serum were collected from 24 unrelated
healthy Dutch subjects. Fresh-frozen sera were stored at -70 °C for
a maximum of 5 months, whereas fresh EDTA blood was used immediately
for DNA isolation. Buffy coat samples and sera that had been stored
frozen for 3–5 years at -20 °C and -70 °C, respectively, were
collected from the Rotterdam Elderly Study (468 samples from unrelated
Caucasians) (7). Low-melting point agarose
plugs (n = 8) that had been genotyped for apo(a) by Trommsdorff et
al. (6) were used for an accuracy check. Cells within these
plugs had been isolated and digested with proteinase K, and the plugs
were used immediately for PFGE. These plugs had been stored in 0.5
mol/L EDTA for 1.5 years prior to their use for PFGE.
serum apo(a) quantification
Serum apo(a) protein was measured using a commercially available
kit (Mercodia). This method is a solid-phase two-site immunoradiometric
assay for which serum samples were diluted 42-fold with a pretreatment
solution. Subsequently, apo(a) was determined in duplicate, using
monoclonal antibodies directed against separate antigenic determinants
on the apo(a) molecule. The procedure was performed according to the
instructions of the manufacturer.
apo(a) genotyping with pfge
Isolation of genomic DNA from fresh blood samples and frozen buffy
coats.
The procedure was based on the method described by
Siraganian and Hook (8). A 3-mL aliquot of fresh EDTA blood,
0.75 mL of dextran/glucose [0.77 mmol/L dextran
(Mr 77 800; Sigma Chemical
Co.) and 166.5 mmol/L glucose in 9 g/L NaCl], and 60 µL of
0.5 mol/L EDTA, pH 8.0, were mixed in a 10-mL sterile conical tube
(Greiner Labortechnik). The tube was placed in a diagonal position of
45 degrees for 15 min and subsequently upright for 60 min for
sedimentation of erythrocytes. The leukocytes remained floating in the
plasma. The plasma was transferred to a new sterile 10-mL tube and
centrifuged (415g for 15 min) at room temperature. The
supernatant was decanted, and the pellet was suspended in 10 mL of
phosphate-buffered saline, pH 7.4. After the sample was
centrifuged at 223g for 7 min at room temperature, the
leukocytes were resuspended in 3 mL of phosphate-buffered saline and a
cell count (Sysmex NE-8000; TOA Medical Electronics) was performed.
After recentrifugation at 223g for 7 min at room
temperature, the leukocytes were resuspended in 25 mmol/L EDTA in 9 g/L
NaCl to a concentration of 3 x 107
cells/mL. The suspension was mixed with two volumes of 10 g/L
low-melting point agarose (Incert agarose; FMC Bioproducts), and the
mixture was aliquoted into disposable plug molds (Bio-Rad). One plug
contained ~7.5 x 105 cells.
For the frozen buffy coats, 250 µL of each sample was transferred
into a 10-mL sterile conical tube (Greiner), and 10 mL of lysis buffer
(20 mmol/L Tris, 10 mmol/L EDTA, pH 8) was added. The tubes were
centrifuged at 3000g for 5 min at room temperature,
and the supernatant was decanted. The sediment was resuspended in the
remaining lysis buffer, 200 µL of 10 g/L low-melting point agarose
was added, and the mixture was poured into disposable plug molds.
After isolation, the plugs were treated with 0.5 g/L proteinase K
(Merck) at 55 °C for 48 h, treated with phenylmethylsulfonyl
fluoride, and stored in 0.5 mol/L EDTA, pH 8.0, according to the
protocol of Trommsdorff et al. (6).
Digestion and PFGE.
Plugs were washed twice with Tris-EDTA
buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 7.5) and once with
Kpn1 digestion buffer (10 mmol/L Tris, 10 mmol/L
MgCl2, pH 7.5) for 30 min. Digestion was carried
out with 2 x 60 U of Kpn1 in a buffer volume of 75
µL in the presence of bovine serum albumin (4 h at 37 °C in 20 g/L
DNA-quality albumin; Boehringer Mannheim). DNA was size separated
(PFGE) with a fragment separation range of 50–175 kb, using the
machine-calculated autoalgorithm according to the protocol of
Trommsdorff et al. (6). Further electrophoresis was
performed with the gel submerged in buffer (50 mmol/L Tris, 50 mmol/L
boric acid, 1 mmol/L EDTA) at 14 °C, and the run time was set at
24 h. As a control, a 
48.5-kb DNA ladder was used (CHEF Mapper
PFGE system, CHEF DNA size standards, Lambda ladder; Bio-Rad).
The gel was stained with ethidium bromide (10 g/L), photographed, and
incubated ~2 min in 10 mL of 4 mol/L NaOH to denature the DNA. The
DNA fragments were blotted (in 0.4 mol/L NaOH overnight at room
temperature) onto a nylon membrane (Boehringer Mannheim) by reversed
capillary blotting using a Turboblotter system (Schleicher & Schuell).
The DNA was fixed by baking the nylon membrane for 30 min at 120 °C.
Probe manufacturing, hybridization, and chemiluminescent
detection.
The probes used for hybridization, directed against
either DNA or apo(a) Kringle IV type 2 repeats, are listed in Table 1
. All probes were prepared with a PCR-digoxigenin-labeling kit
(Boehringer Mannheim). Template DNA for the apo(a) probes was prepared
by PCR reaction on genomic DNA with all four apo(a) primer sets. PCR
was performed in a 110-µL reaction volume with 10 µL of buffer (0.5
mol/L KCl, 0.1 mol/L Tris, 25 mmol/L MgCl2, pH
9.0, containing 1 g/L gelatin and 10 mL/L Triton X-100), 20 µL of 1
mmol/L dNTP, 1.2 µL of primer (50 pmol/µL Lp1–Lp4), 0.3 µL of
Promega Taq (5 U/µL), 67 µL of sterile water, and 10 µL of
genomic DNA. The reaction mixture was overlaid with mineral oil. The
PCR conditions were as follows: first denaturation step, 7 min
at 95 °C; followed by 40 cycles of denaturation for 45 s
at 95 °C, annealing for 1 min at 60 °C, and elongation for 2 min
at 72 °C. The final elongation step was 7 min at
72 °C.
Template DNA was purified using microspin columns (QIAquickspin PCR
Purification kit; Qiagen) according to the manufacturer's protocol.
With this template solution, a PCR reaction was performed in a 110-µL
reaction mixture containing 1:5 digoxigenin-dUTP:dTTP; 10 µL of 0.5
mol/L KCl in 0.1 mol/L Tris, pH 8.3; 5 µL of synthesis mix (2 mmol/L
dATP/dCTP/dGTP, 1.3 mmol/L dTTP, 0.7 mmol/L digoxigenin-11-dUTP), 5
µL of 2 mmol/L dNTP stock solution, 1.2 µL of primer (50
pmol/µL), 1.5 µL of Taq expand mix (1 U/µL), 76 µL of water,
and 10 µL of template DNA. The reaction mixture was overlaid with
mineral oil. The cycle conditions were as follows: a first denaturation
step for 2 min at 95 °C; followed by 10 cycles of
denaturation for 10 s at 95 °C, annealing for 30 s at
60 °C, and elongation for 2 min at 72 °C; and 20 cycles of
denaturation for 10 s at 95 °C, annealing for 30 s at
60 °C, and elongation for 2 min at 72 °C, with a 20-s time
extension for the elongation step in each cycle of the last 20 cycles.
The final elongation step was for 7 min at 72 °C. After
electrophoresis of 10 µL of the PCR product, the product band was
excised from the gel, and the labeling efficiency was checked by dot
spotting of a dilution series. We used the lowest detectable dilution
fraction as the basis for choosing the amount of labeled probe to be
purified for comparison with previous labeling experiments (usually
6–8 µL). Hybridization was carried out overnight at 42 °C in the
presence of 500 mL/L formamide, according to the method of Sambrook et
al. (9). Chemiluminescent detection of the apo(a) genotype
was performed according to the Digoxigenin System user's guide
(Boehringer Mannheim). Alkaline phosphatase-conjugated
anti-digoxigenin-Fab fragments were used as the antibody-conjugated
enzyme for the chemiluminescence reaction. CSPD®
[disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo
[3,3,1,13,7]-decan}-4-yl)phenyl
phosphate; Boehringer Mannheim] was used as the substrate for
alkaline phosphatase. Gel images were made using Kodak Biomax-MR films;
the first image of every gel was made when steady state was reached
(60 min after addition of the substrate). Three exposures were
made of each gel, with exposure times of approximately 15, 30, and 45
min.
Reproducibility and precision.
We prepared a control sample
for imprecision studies by combining leukocytes (fresh samples) of four
selected heterozygous volunteers, which yielded eight apo(a) fragments
that covered the entire fragment size range of 72–233 kb. The combined
cell count of the control sample was increased to 2 x
107/mL in the plugs to ensure adequate detection
signals for all eight apo(a) fragments. Plugs were digested with
proteinase K and stored (4 °C) in 0.5 mol/L EDTA, pH 8. DNA
digestion was performed with 2 x 120 U of Kpn1 before
electrophoresis.
calculation of the apo(a) genotype
Kpn1 digestion of genomic DNA yields apo(a) fragments
containing all Kringle IV (type 2) repeats as well as four nonrepeating
Kringles (types 1, 3, 4, and 5). The size of the fragments after these
four nonrepeating Kringles (29.932 kb) are subtracted equals the number
of Kringle IV type 2 repeats. Five Kringle IV repeats (types 6–10)
remain in the genomic DNA; therefore, the apo(a) genotype is calculated
by adding the repeating type 2 Kringles to all nonrepeating Kringles
(nine Kringles). The films were scanned, and final quantification was
performed using Gelcompar 3.0/4.0 (Applied Maths BVBA). The DNA
marker (48.5-kb Lambda ladder; Bio-Rad) was used as a reference for
fragment size calculation as well as for correction of gel-to-gel
variation by the Gelcompar normalization program. All Kpn1
fragments were identified by interpolation of migration distances,
using an exponential curve fitting. Calculations of the apo(a) genotype
were based on the work by Lackner et al. (10) and the
information presented by the National Center for Biotechnology
Information GenBank database (GenBank name, HUMAPOAKIV; GenBank
accession no. L14005; se- quence ID 806604:1.5533). The
formulas are shown below, with one Kringle IV repeat representing 5.533
kb:
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Results
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optimization of the apo(a) genotyping procedure
The dextran procedure enables the isolation of all leukocytes from
fresh blood samples; the isolation of all leukocytes from frozen buffy
coats was accomplished by a centrifugation method. The DNA yield of
both isolation methods allowed for comparable analytical sensitivity
(Fig. 1
). When the centrifugation method was used on frozen
EDTA whole blood, a less intense detection signal was found despite the
fact that a comparable number of leukocytes (7.5 x
105) were present in the plug (Fig. 1A
, lane 7).
An extra wash step with digestion buffer was introduced before
Kpn1 digestion of isolated DNA. DNA samples digested
according to existing protocols (Fig. 2
A) were compared with the modified method (Fig. 2B
). Digestion
efficiency was increased in the modified method, as visualized
by the disappearance of the high-molecular weight DNA band in the
non-resolution zone. Reversed capillary blotting was used for
transferring DNA from the gel to a nylon membrane. Visibly empty gels
were obtained, indicating complete DNA transfer. Increased sensitivity
was further accomplished by the use of four different apo(a) probes.
Improved accuracy and practicality were obtained by the addition of a
marker probe in the same hybridization mix, enabling detection on a
single film (see Fig. 1
; markers are in the outer lanes).
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Figure 1. Effect of sample type and isolation procedure on the
sensitivity of the PFGE apo(a) genotyping procedure.
(A), photographic film of apo(a) genotypes from fresh
EDTA blood (except lane 7); (B),
photographic film of apo(a) genotypes from frozen buffy coats. A strong
detection signal was found with either sample type, indicating adequate
sensitivity for the apo(a) genotyping procedure. (A),
lanes 1 and 11, Bio-Rad 48.5-kb Lambda
ladder; lane 7, apo(a) genotype from a frozen EDTA blood
sample (kept at -70 °C for 7 days); (B),
lanes 1 and 10, Bio-Rad Lambda 48.5-kb
ladder; lane 2, human control sample.
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Figure 2. Effect of an extra wash step with digestion buffer.
(A), negative of an ethidium bromide-stained gel, after
PFGE, that did not undergo an extra wash step with digestion buffer.
(B), negative of an ethidium bromide-stained gel, after
PFGE, that underwent an extra wash step with digestion buffer. The
increased Kpn1 digestion efficiency is illustrated by
the decrease in high molecular weight DNA fragments in the
non-resolution zone in B. The Bio-Rad 48.5-kb Lambda
ladder and New England Biolabs midrange I/II Lambda markers were used.
(A), from left to right,
lanes 1, 7, and 15,
midrange II Lambda markers; lanes 2 and
19, Bio-Rad Lambda ladder; lanes 3,
11, and 20, midrange I Lambda markers;
lanes 4–6, 8–10, 12–14,
and 16–18, DNA samples. (B), from
left to right, lanes 1 and
14, midrange II Lambda markers; lanes 2,
6, 18, and 20, Bio-Rad
Lambda ladder; lanes 10 and 19, midrange
I Lambda markers; lanes 3–5, 7–9,
11–13, and 15–17, DNA samples. The
non-resolution zone is indicated.
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validation
The reproducibility of the modified PFGE method was determined
with the control sample, obtained by combining leukocytes from four
volunteers (as described in Materials and Methods).
Kpn1 fragment size, as determined over 34 separate
electrophoretic runs, was reproducible to within one or fewer Kringle
IV repeats, with an SD 
2.5 kb and a CV 3%. To ensure accuracy, the
results were accepted only when the apo(a) genotype determination of
the eight control fragments was within one Kringle IV repeat of the
established value (within first electrophoretic run, n = 3).
Furthermore, the accuracy of the method in relation to the absolute
number of Kringle IV repeats was verified on DNA samples that were
genotyped by Trommsdorff et al. (6). One of the eight
samples obtained could not be genotyped because of denaturation of the
DNA, and a smear was seen in the gel and on the blot. All 14
genotyped apo(a) fragments differed by no more than one Kringle IV from
the results of Trommsdorff et al. (6).
relationship between apo(a) genotype and serum Lp(a) concentrations
An inverse correlation between serum Lp(a) concentrations and the
number of Kringle IV repeats in either allele was found. When serum
Lp(a) concentrations were plotted against the sum of the Kringle IV
repeats in both alleles, a negative correlation was found
(y = -22.757x + 1553.3; r =
-0.442). From the frozen buffy coat samples tested (n = 468),
93% were found to be heterozygous for the apo(a) Kringle IV repeat
size.
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Discussion
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The rationale of this study was to improve both the sensitivity
and practicality of currently described apo(a) genotyping procedures.
Overall sensitivity of the procedure can be increased by optimization
of each step in the procedure, starting with the leukocyte isolation.
The availability of tiny blood samples or frozen buffy coats persuaded
us to search for a modified isolation procedure in which a high DNA
yield allowed the reduction of sample volume. We selected a dextran
method and a centrifugation method, which isolated all leukocytes from
the blood samples and buffy coats, respectively. In contrast to the
isolation of mononuclear cells, this modification theoretically
produces a threefold amplification of the DNA yield. Dextran
accelerates sedimentation of erythrocytes by "rouleau" formation(11), and increased plasma density keeps leukocytes floating
in the plasma layer. The recovery of leukocytes in the dextran
isolation method was not fully reproducible between samples. It was
necessary to perform a cell count after the dextran isolation to ensure
the presence of 7.5 x 105 cells in the
plugs. Because the freezing process damages and clusters white blood
cells (and DNA), a cell count could not be performed when the frozen
buffy coats were used. We started with the same sample size for all
frozen buffy coats and concentrated the isolated leukocytes (DNA) into
the plugs as much as possible, which produced a certain variation in
detection signal. Notwithstanding, the proposed procedure enabled us to
determine apo(a) genotypes in buffy coats that had been kept frozen at
-20 °C for 3–5 years.
Traces of EDTA, which were present in the plugs after the Tris-EDTA
buffer wash, captured magnesium ions, an essential cofactor of
Kpn1. For complete digestion by Kpn1, it was
necessary to introduce an extra wash with digestion buffer to saturate
the remaining traces of EDTA with magnesium. With this modification, an
optimal magnesium concentration in the Kpn1 digestion
reaction was ensured and led to efficient cutting of the genomic DNA. A
higher yield of each separated apo(a) fragment was obtained, and a
stronger detection signal and improved sensitivity were found.
Reversed capillary blotting enabled easy-to-perform, time-saving
stack-building, which yielded highly reproducible results.
The final step taken to increase the detection signal was the
combination of four different apo(a) probes, which enhanced the
detection signal. The probes were designed to hybridize within one
Kringle IV type 2A repeat. Hybridization sites are distributed over the
length of the two introns and two exons. However, the detection signal
may increase only proportionally because the increase in the number of
apo(a) probes that can be hybridized is limited by steric hindrance
from the incorporated digoxigenin label in the probes. Integrated
detection of markers and unknown samples was accomplished by the
addition of a labeled marker probe to the hybridization mixture of
the apo(a) probes. This procedure allowed for single film detection,
which improved accuracy and practicability because markers and unknown
samples were visible on the same film. The sensitivity of detection
(with all probes) was influenced most strongly by the amount of
digoxigenin label incorporated in the probes by PCR labeling. From a
standardization viewpoint, it is therefore essential that the amount of
labeled probe administered to the hybridization mix is kept at a
constant concentration throughout the entire investigation.
Published procedures (5)(6) indicate that
discrimination should be possible for single apo(a) Kringle IV
repeats. This is in agreement with our findings. Furthermore,
the modified method was reproducible to within one or fewer Kringle IV
repeats. If fragments of the control sample differ by more than one
Kringle IV repeat from the assigned value, repeated analysis of the
samples is required. This happened only once throughout the whole
investigation.
Analogously to Lackner et al. (5), we found an inverse
relationship between serum Lp(a) concentration and the number of
Kringle IV repeats in either allele. In our epidemiological
study, a heterozygosity index of 93% was found, which is comparable to
the 94% of Lackner et al. (10). The results of our
epidemiological study of Caucasians, in which we used buffy coats
stored frozen at -20 °C for several years, were comparable to the
results of the studies using fresh EDTA blood samples(5)(10).
In summary, the proposed modifications improved the apo(a)
genotyping procedures described to date and made them suitable for
repeated analysis with small blood sample volumes and with frozen buffy
coats. Because the DNA isolation, Kpn1 digestion,
hybridization, and detection steps were optimized, a difference of one
or fewer Kringle IV repeats in apo(a) could be detected. When 3 mL of
EDTA blood or 250 µL of buffy coat is used as the starting material,
adequate sensitivity can be achieved with the use of four different
apo(a) probes. These modifications, when used together, make the apo(a)
genotyping procedure suitable and practical for large-scale apo(a)
genotyping studies such as large epidemiological projects and clinical
trials.
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Acknowledgments
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This work was funded by the Dutch Heart Foundation (Grant 94.032).
We thank Dr. Y.Y. van der Hoek for arranging the exchange of the DNA
plugs with defined apo(a) genotypes. We thank Prof. Dr. G. Utermann and
Prof. Dr. H.G. Kraft of the Institute for Medical Biology and Human
Genetics, Innsbruck, Austria, for the apo(a) genotyping of the plugs
and for allowing us to use the data for the accuracy check.
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Footnotes
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1 Nonstandard abbreviations: Lp(a), lipoprotein a; apo(a), apolipoprotein a; and PFGE, pulsed field gel electrophoresis. 
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Abstract
Introduction
