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Clinical Chemistry / APOLIPOPROTEIN E ALLELE DISTRIBUTION IN TRISOMY 13, 18, AND 21
Apolipoprotein E Allele Distribution in Trisomy 13, 18, and
21 Conceptuses in a Hungarian Population
Bálint Nagy, PhD,1 Zoltán Bán, MD,1 Ernö Tóth-Pál, MD,1 Csaba Papp, MD,1
Lou Fintor, MA, MPH,2 and Zoltán Papp, MD, PhD, DSc1
Key Words: Fluorescent polymerase chain reaction; Trisomy 13; Trisomy 18; Trisomy 21; Apolipoprotein E
Abstract
Reports documented a higher frequency of
apolipoprotein E (apoE) allele e4 among mothers of
children diagnosed with Down syndrome. We studied
the prevalence of apoE alleles among 56 conceptuses
with trisomy 13, trisomy 18, or trisomy 21. The
presence of the 3 most common apoE alleles (e2, e3,
e4) was determined by polymerase chain
reaction–restriction fragment length polymorphism, and
trisomy status was detected by fluorescent polymerase
chain reaction followed by DNA fragment analysis and
by conventional cytologic methods. We found no
significant difference in the distribution of apoE alleles
in the group of trisomy 21 fetuses compared with
samples from healthy blood donors. The odds of having
trisomy 18 for the apoE e4 group was 3-fold as high as
for apoE e3 allele compared with the healthy control
group. Furthermore, a statistically significant
association was found for those with trisomy 18 and
apoE e4, while for those with trisomy 13 and apoE e4,
the test showed no significant association. The observed
apoE allele e3 frequencies among patients with Down
syndrome and healthy control subjects may help explain
and support previous work that did not find high rates
of atherosclerosis among these persons. The role of
apoE alleles in the development of trisomies needs
further study.
© American Society of Clinical Pathologists
Chromosomal abnormalities are the most frequent genetic
disorders observed in live births and miscarriages; more than
90% of these cases are attributable to numeric variations of
chromosomes 21, 18, and 13.1 These variations usually are
diagnosed via in vitro cultures of nucleated cells followed by
standard karyotyping procedures or through fluorescence in
situ hybridization.2 Fluorescent polymerase chain reaction
PCR (F-PCR) recently has been used more commonly for the
detection of trisomy conditions.3-5
Intrauterine mortality remains very high among cases of
genetic trisomy; in the case of trisomies 13 and 18, only 5%
of the cases results live birth, and most of these infants
usually die within the first year of life. Among trisomy 21
conceptuses, approximately 15% of the cases result in live
birth, and the first-year mortality rate also is quite high. In
addition, of the children who survive the first year of life,
respiratory infections, cardiac conditions, or leukemia
develop in many. With proper treatment, however, many live
beyond age 50 years and some well into their 60s. Among
patients diagnosed with the nondisjunction of chromosome
21 (trisomy 21) that is commonly referred to as Down
syndrome, investigators have documented high mortality
rates but also a low incidence of atherosclerosis that persists
even through the fifth and sixth decades of life.6 For example,
Yla-Herttuala et al7 found a significantly lower number of
raised lesions and less calcium in the coronary arteries of 15
persons with Down syndrome. Nevertheless, there is little
consensus in the literature about lipid marker levels found in
these patients in the absence of genetic studies. Boccini et al6
studied the cholesterol and triglyceride levels in 18 fetuses
with trisomy 21 and found higher levels of these lipids in
utero compared with those with normal karyotypes. They
concluded that increased triglyceride levels might be related
Am J Clin Pathol 2000;113:535-538
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Nagy et al / APOLIPOPROTEIN E ALLELE DISTRIBUTION IN TRISOMY 13, 18, AND 21
to some degree of placental impairment rather than to the
genetic abnormality itself. Meanwhile, Brattstrom et al8
found higher cystathionine synthase activity among patients
with Down syndrome that they attributed to a higher expression of the gene. However, apolipoprotein E (apoE) is an
important factor in lipid metabolism. Synthesized mostly in
the liver, apoE alleles are established genetic markers for
dyslipidemia and coronary heart disease.9,10
Apolipoprotein E allele frequencies among fetuses with
a trisomy condition have not been studied and reported.
Materials and Methods
DNA Preparation and Cytogenetic Analysis
Amniotic fluid specimens were collected from women
participating in the Semmelweis University Medical School
(Budapest, Hungary) genetic screening program. From
January 1997 through December 1998, 56 samples were
trisomic. The average age of women with a trisomic fetus
was 34.2 years, and the mean gestational age was 15.1
weeks. Institutional review board approval was sought and
granted from Semmelweis University, a national maternal
health referral center, and informed consent was obtained
from all study participants.
Amniotic fluid samples of 1.5 mL were centrifuged at
15,000 rpm for 10 minutes to obtain amniocytes, and the
supernatant was discarded. Resin, 100 µL (ReadyAmp
Genomic Purification System, Promega, Madison, WI),
was added to the pellet, and DNA extraction was performed
according to the manufacturer’s standard protocol. Four
microliters of this DNA solution was used in each PCR
reaction. Cytogenetic analysis was performed according to
established protocol.11
Fluorescent PCR
PCR amplification was performed to detect chromosomal
numeric abnormalities using primer pairs D21S11, D21S1411,
D21S1412, D13S631, D13S258, D18S851, and D18S51. The
forwarding primer was fluorescent dye labeled in each set, and
the exact composition of the PCR reaction was identical to that
used previously.4,5
Separation and Quantitation of F-PCR Products
Four microliters of F-PCR product was mixed with 24
µL of formamide (Amresco, Solon, OH) and 2 µL of Prism
Genescan-500 TAMRA size standard (PE Applied Biosystems, Warrington, Great Britain). The mixture was denatured at 95 C for 3 minutes and placed on ice for 5 minutes
followed by electrophoretic analysis using POP4 gel (PE
Applied Biosystems, Foster City, CA). Amplification products
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Am J Clin Pathol 2000;113:535-538
were analyzed using Genscan Analysis 2.1 software (PE
Applied Biosystems).
ApoE Allele Determination With PCR–Restriction
Fragment Length Polymorphism
ApoE genotyping was performed with 4 µL of isolated
DNA (from amniotic fluid) via PCR–restriction fragment
length polymorphism (RFLP) according to standard and
accepted protocols.9,10,12,13 ApoE alleles were determined after
visualization of DNA fragments through HhaI digestion. Blood
samples from healthy donors also were collected for DNA
extraction and used as controls (n = 253; mean age, 39.9 years).
Statistical Analysis
Statistical analysis was performed using SPSS software
(Statistical Package for the Social Sciences; SPSS, Chicago,
IL) to calculate all odd ratios and establish confidence intervals (CIs) at the 95% threshold. Statistically significant associations reported at the P < .05 threshold were determined using
the Fisher exact test, and accompanying values were reported
as statistically significant only when they were P < .05.
Results
Trisomy status was detected using conventional cytologic
methods along with F-PCR followed by DNA fragment
analyses. Results were obtained consistently using both aforementioned methods. In all trisomic samples, apoE alleles were
assigned using PCR-RFLP.
Among the 56 trisomy cases, 33 patients (59%) were
homozygous for apoE e3, whereas among healthy control
subjects, 167 (66.0%) were homozygous for e3 ❚Table 1❚.
In the trisomy 18 group, which comprised a total of 34
alleles, 7 apoE e4 alleles (20%) and 23 apoE e3 alleles (68%)
were detected. To study the potential association between
trisomy type and apolipoprotein, alleles were isolated from the
samples of 253 healthy blood donors. Of the resulting 506
healthy control alleles, 413 apoE e3 alleles (81.6%) were
❚Table 1❚
Distribution of Apolipoprotein E Genotype in Conceptuses
Showing Trisomy Compared With Samples From Healthy
Control Subjects*
Genotype
E 2/2
E 2/3
E 2/4
E 3/3
E 3/3
E 4/4
Trisomy Group (n = 56)
0 (0)
8 (14)
2 (4)
33 (59)
12 (21)
1 (2)
Control Group (n = 253)
5 (2.0)
41 (16.2)
2 (0.8)
167 (66.0)
37 (14.6)
1 (0.4)
* Data are given as number (percentage).
© American Society of Clinical Pathologists
Clinical Chemistry / ORIGINAL ARTICLE
found. By considering apoE e3 as a common “normal” allele,
we computed odds ratios and a 95% CI by comparing each
trisomy type with the healthy control group ❚Table 2❚.
A statistically significant association between apolipoprotein and trisomy status was determined based on a 95% CI and
odds ratio using the Fisher exact test. The only CIs that
excluded 1 were the groups that combined apoE e4 and
trisomy 13 and the group with trisomy 18 and apoE e4 (Table
2). Thus, the odds of having trisomy 18 for those in the apoE
e4 group is 3-fold as high as apoE e3 compared with healthy
controls (95% CI, 1.27-7.43). Furthermore, the test showed a
statistically significant association for those with trisomy 18
and apoE e4 (P < .05), while for those with trisomy 13 and
apoE e4, the test showed at least some significant association
(P = .07)
Discussion
Chromosomal aneuploidy remains one of the major
causes of pregnancy wastage in humans. While increased
maternal age has been established as a risk factor for chromosomal abnormalities, the biologic mechanism of this phenomenon remains unknown.14 Trisomies 21, 18, and 13 are the
most frequently observed aneuploidy conditions among
newborns.
Lately, apoE and its alleles have become the focus of
investigations.14-17 Avramopoulos et al14 found a higher
frequency of the apoE e4 allele in young mothers of children
with Down syndrome with a meiosis II error. They suggested
that this allele is a risk factor,14 while the biologic role of apoE
in oocytes remains to be studied. Hansen et al18 studied the
frequency of apoE alleles in a combined cohort of samples and
found higher e2 allele frequencies among mothers of trisomy
18 conceptuses with meiosis I errors. They combined samples
from different countries, but it is important to note that ethnic
differences exist in the distribution of apoE alleles.
There are similarities in Alzheimer disease and Down
syndrome. Hymen et al19 studied the epidemiologic differences
associated with the apoE genotype. In both groups of patients,
increased beta-amyloid protein deposition was observed in the
cerebral lesions and cerebral blood vessels. They found a
higher frequency of apoE e4 alleles in patients with Alzheimer
disease, while in patients with Down syndrome, the frequency
did not differ from that of the healthy control group. The mechanism of the deposition of senile plaques seems to be different.
In trisomy 21, there are large plaques reflecting increased betaamyloid production, probably due to the higher activity of the
amyloid gene, which is located in the chromosome 21q22
region. This region seems to be critical in the pathogenesis of
the Down syndrome phenotype.19
We studied the frequency of apoE alleles among conceptuses with trisomy 21, 18, or 13 in ethnically homogeneous
Hungarians. We examined DNA samples of fetuses around the
15th week of gestation so we could include the cases that
might not reach full-term gestation period. A healthy population from same region was used as the control group. We
applied 2 methods for the detection of aneuploidies, the
conventional cytogenetic and F-PCR followed by DNA fragment analysis. PCR-RFLP was used for apoE genotyping;
DNA fragments were separated on polyacrylamide gels and
visualized with silver staining. We found a higher frequency of
apoE e4 allele in the trisomy 13 and trisomy 18 groups. In the
trisomy 21 group, we did not observe statistically significant
differences in the distribution of apoE alleles. This finding is
in accordance with those of previous studies on smaller
numbers of patients with Down syndrome.20,21
The biologic role of apoE in the oocyte and the role of the
e4 allele in the development of aneuploidies remain to be
studied. In the case of trisomy 13 and 18, the microcirculation
theory could have a role in the pathogenesis according to
Avramopoulos et al.14 As e4 allele carriers have higher plasma
cholesterol levels, which is related strongly to atherosclerosis,
it could lead to an oxygen deficit inside the follicle and, in
turn, diminish the size of the spindle with consequent nondisjunction of a chromosome.14 For trisomy 13 and 18, our
results support this explanation.
Another hypothesized mechanism for chromosomal
❚Table 2❚
Distribution of Apolipoprotein E (ApoE) Alleles for Conceptuses Showing Trisomy 13, 18, or 21 and in Samples From Healthy
Control Subjects*
Allele
ApoE e2
Conceptuses showing trisomy
13 (n = 18)
18 (n = 34)
21 (n = 60)
Healthy control alleles (n = 506)
*
†
1 (6)
4 (12)
5 (8)
52 (10.3)
ApoE e4
4 (22)
7 (20)†
5 (8)
41 (8.1)
ApoE e3
13 (72)
23 (68)
50 (83)
413 (81.6)
Data are given as number (percentage).
P < .05.
© American Society of Clinical Pathologists
Am J Clin Pathol 2000;113:535-538
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Nagy et al / APOLIPOPROTEIN E ALLELE DISTRIBUTION IN TRISOMY 13, 18, AND 21
segregation is the allele-specific antioxidant activity protecting
from oxidative cell death, with E2 being the most protective
followed by E3 then E4.16,18,22 The 2-hit model also is in use,
and apoE could represent an example of disruption of the
meiotic process due to the isoform-specific binding to microtubule-associated proteins and possible interference with
microtubule stability and function.18,23,24 It is difficult to
decide which is the most appropriate of the aforementioned
theories.
We conclude that the frequency of the e4 allele is higher
in the case of trisomies 13 and 18. We found no difference in
the case of trisomy 21 compared with the healthy Hungarian
population. This observation helps explain and support the
findings of the studies that did not find significantly higher
rates of atherosclerosis among patients with Down
syndrome.7,20,21 It also supports the hypothesis that suggests
more cystathione synthetase production (the gene is located on
chromosome 21) and as a consequence, lower homocysteine
levels in these patients. These preliminary results require additional study to unravel the elusive cause of chromosomal
numeric abnormalities.
From the 1Molecular Diagnostics Laboratory, First Department of
Obstetrics and Gynecology, Semmelweis University Medical
School, Budapest, Hungary; and the 2Department of Internal
Medicine, The University of Michigan Medical School, Ann Arbor,
MI.
Mr Fintor is now with McGill University, Montreal, Quebec,
Canada.
Address reprint requests to Dr Nagy: Liljasaarentie 3A2,
00340 Helsinki, Finland.
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© American Society of Clinical Pathologists