Apolipoprotein E gene polymorphism and polycystic ovary syndrome patients in Western Anatolia, Turkey.

Abstract

Dyslipidemia, cardiovascular disease and hypertension are more frequently seen in patients with PCOS than in normal patients. We aimed at evaluating the distribution of Apo E alleles that can influence cardiovascular risk of the PCOS patients and control subjects.
In this study, 129 young women with PCOS and 91 healthy women were included. In all subjects we performed hormonal, biochemical and Apo E genetic analysis.
The Apo E3 allele was found at a significantly higher frequency in the PCOS patient group compared with the control group. The Apo E2 allele was found at a significantly higher frequency in the control group compared with the patient group with PCOS.
Although there were genotype and allele differences between control and patient groups in this study, no statistically significant change was determined in lipid and other cardiovascular risk factors in connection with allele and genotype.

Full text

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AROUND THE WORLD
Apolipoprotein E gene polymorphism and polycystic ovary
syndrome patients in Western Anatolia, Turkey
Sevki Cetinkalp & Muammer Karadeniz &
Mehmet Erdogan & Ayhan Zengi & Vildan Cetintas &
Aslı Tetik & Zuhal Eroglu & Buket Kosova &
A. Gokhan Ozgen & Fusun Saygili & Candeger Yilmaz
Received: 19 June 2008 /Accepted: 13 November 2008 /Published online: 5 December 2008
# Springer Science + Business Media, LLC 2008
Abstract
Purpose Dyslipidemia, cardiovascular disease and hyper-
tension are more frequently seen in patients with PCOS
than in normal patients. We aimed at evaluating the
distribution of Apo E alleles that can influence cardio-
vascular risk of the PCOS patients and control subjects.
Methods In this study, 129 young women with PCOS and 91
healthy women were included. In all subjects we performed
hormonal, biochemical and Apo E genetic analysis.
Results The Apo E3 allele was found at a significantly
higher frequency in the PCOS patient group compared with
the control group. The Apo E2 allele was found at a
significantly higher frequency in the control group com-
pared with the patient group with PCOS.
Conclusions Although there were genotype and allele
differences between control and patient groups in this
study, no statistically significant change was determined in
lipid and other cardiovascular risk factors in connection
with allele and genotype.
Keywords ApolipoproteinEgene.Cardiovascularrisk
factors.Hyperlipidemia.Polycysticovarysyndrome
Abbreviations
ApoE
PCOS
BMI
CI
CV
MLRA
CVD
DHEAS
E2
Hcy
17-OHP
P
PRL
T
FSH
LH
HOMA-IR
Apolipoprotein E
polycystic ovary syndrome
body mass index
confidence interval
coefficient(s) of variation
Multiple Logistic Regression Analysis
cardiovascular disease
dehydroepiandrosterone sulphate
17ß-estradiol
homocysteine
17-hydroxyprogesterone
progesterone
prolactin
testosterone
follicle stimulating hormone
luteinizing hormone
homeostasis model of assessment
insulin resistance
Introduction
Polycystic ovary syndrome (PCOS) is one of the most
frequent endocrine malfunctions, which typically occur
with chronic anovulation and hyperandrogenism [1].
Almost 7% of women at reproductive age are diagnosed
with PCOS [2].
Dyslipidemia, cardiovascular disease and hypertension
are more frequently seen in patients with PCOS than in
normal patients [3–5].
The disorder has also been reported to be associated with
an increase in sub-clinical atherosclerotic disease [5].
J Assist Reprod Genet (2009) 26:1–6
DOI 10.1007/s10815-008-9280-8
S. Cetinkalp:M. Karadeniz (*):M. Erdogan:A. Zengi:
A. G. Ozgen:F. Saygili:C. Yilmaz
Division of Internal Medicine,
Department of Endocrinology and Metabolism Disease,
Ege University Hospital,
Izmir 35100, Turkey
e-mail: muammerkaradenis@gmail.com
V. Cetintas:A. Tetik:Z. Eroglu:B. Kosova
Department of Medical Biology, Ege University Hospital,
Izmir, Turkey

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Apolipoproteins that form the protein part of the lipo-
proteins are important in lipid metabolism. It regulates the
transport and redistribution of lipoproteins in blood as a
cofactor. Apolipoprotein E (ApoE) is a 34,200 kDa
polymorphic glycoprotein consisting of 299 amino acids
[6]. Three common alleles of ApoE are E2, E3, E4 code for
three isoforms E2, E3 and E4 in exon 4 ApoE genes. These
isoforms result in six common phenotypes, which are E2/2,
E2/3, E2/4, E3/3, E3/4 and E4/4 [7]. Relation of ApoE2 to
the threat of atherosclerosis is controversial. ApoE4 is
connected with decreased longevity, increased plasma lipid
and ApoB levels and higher prevalence of cardiovascular
illness and also of Alzheimer’s [8, 9].
Addition of genetic risk factors (such as abnormality of
Apo E, PPAR, and TPA-PAI) to increasing technology and
inactivity increases the human susceptibility to cardiovas-
cular disease in the future. To date, there is no data
available for Turkish subjects in the genetic polymorphism
of ApoE and its relation with increased cardiovascular risk
factors in PCOS patients. In this study, we aimed at
evaluating the distribution of ApoE alleles that can
influence cardiovascular risk of the Turkish PCOS patients
and control subjects.
Materials and methods
Patients
In this study, 129 young women with PCOS and 91 healthy
women were included. PCOS was defined by Rotterdam
PCOS consensus criteria [10].
Patients who had Diabetes Mellitus (diagnosed by 75 g
Oral Glucose Tolerance Test), hyperprolactinemia, congen-
ital adrenal hyperplasia (diagnosed with the adrenocor-
ticotropic hormone stimulation test), thyroid disorders,
Cushing’s syndrome, hypertension, hepatic or renal dys-
function were excluded from the study. At study entry, all
subjects underwent venous blood drawing for complete
hormonal assays, lipid profile, glucose, and insulin. All
blood samples were obtained during early follicular phase
of the spontaneous or progesterone-induced menstrual cycle
in the morning between 8 AM and 9 AM respectively after an
overnight fasting, and while resting in bed.
Biochemical assay
Serum concentrations of hs-CRP were determined by an
immunonephelometric assay (N-hs-CRP; Dade Behring,
Izmir, Turkey); intra- and inter-assay CVs were 1.72 and
2.80%, respectively. Serum total cholesterol, LDL and HDL
cholesterol, triglyceride, aspartate aminotransferase (AST),
alanine aminotransferase (ALT), and -glutamyltransferase
(GGT) were measured by Olympus AU 2,700 automated
analyzer (Toshiba, Tokyo, Japan). Plasma insulin con-
centrations were determined by Immulite 2000 that uses
two-site chemiluminescent immunometric assay. Insulin
resistance was calculated by using homeostasis model
assessment insulin resistance index (HOMA-IR) [11]
according to the following formula:
HOMA?IR
¼fastingseruminsulin mU=ml
ð Þ ? fastingplasmaglucose mmol=l
22:5
ðÞ
ApoE gene genotyping
Genomic DNA was extracted from peripheral leukocytes of
the subjects using the High Pure PCR Template Preparation
Kit (Roche Applied Science). For the detection of the
presence of the three ApoE, E alleles E2, E3 and E4 (codon
112 and 158) were analyzed by the commercial LightCycler
ApoEMutationDetectionKit(RocheDiagnostics,Mannheim,
Germany).
All experiments were carried out on LightCycler™
Instrument (Roche Applied Science, Germany) according
to the protocols provided by manufacturer. Polymorphic
alleles were identified by specific melting temperature (Tm)
of the resulting amplicons. In the case of ApoE, codon 112
and 158 were analyzed simultaneously and therefore two
Tm values were obtained for each allele: for E2, these were
56°C and 57.5°C; for E3, 56°C and 66°C; and for E4, 62.5°C
and 66°C respectively.
Statistical analysis
SPSS 14.0 for Windows (SPSS Inc. Chicago, USA) was
used for statistical analysis of the results. P<0.05 values
were accepted as statistically significant. The characteristics
of the patients with PCOS and the mean plasma homo-
cystein, glucose, and insulin, dehydroepiandrosterone sul-
phate, 17ß-estradiol, homocystein, 17-hydroxyprogesterone,
prolactin, testosterone levels between the two clinical groups
were compared by Student’s t test for unpaired data and
between and within the different groups (E2/E3, E3/E3, E3/
E4) of ApoE genotypes with the ANOVA. To determine
which of the parameters, for which Student’s t test found a
statistically significant difference between patient and control
groups, was more strongly associated with PCOS, we
performed univariate logistic regression analysis. Differences
in the genotype distribution between different groups were
assessed by logistic regression analysis of heterogeneity. All
results were expressed as means ± SD.
2 J Assist Reprod Genet (2009) 26:1–6

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Results
Biochemical and hormonal parameters of the patients with
PCOS and the control group are seen in Table 1. The levels
of total cholesterol (p=0.004), LDL-cholesterol (p=0.023),
triglyceride (p=0.004), fasting glucose (p<0.001), fasting
insulin (p<0.001), homocystein (p<0.001), fibrinogen (p<
0.001), FSH, LH, 17-OHP and free-testosterone (p<0.001)
in patients with PCOS were determined to be statistically
significantly high. Statistically significant difference was
not determined for age and BMI between both groups.
Again, no statistically significant difference was determined
between control and patient groups for DHEAS, estradiol,
total testosterone, prolactin, HDL-cholesterol, and hs-CRP
levels.
ApoE genotype distribution in patient and control groups
is seen in Table 2. There is no patient or healthy subject
with E2/E2 and E4/E4 genotypes in our study. E2/E3 and
E3/E3 genotypes were determined to be higher in control
group (p<0.001) and E3/E3 was determined to be higher in
the group of patients with PCOS (p<0.001, OR: 6.34, CI:
2.93–13.74). However, E3/E4 genotype was determined to
be at similar levels in patient and control groups (p=0.07,
OR: 1.94, CI: 0.67–5.66). The ApoE3 allele showed a
significantly higher frequency in PCOS patient group
(91.8%) when compared with control group (75.6%) (p<
0.001). The ApoE2 allele showed a significantly higher
frequency in the control group (16.7%) when compared
with patient group with PCOS (4.3%) (p<0.001). In this
study, ApoE4 allele frequency was not statistically signif-
icantly different in patient and control groups (p>0.513).
Metabolic and hormonal levels were analyzed in patients
with PCOS according to different allele situations (E2, E3,
E4) of Apo E. (Table 3). As a result of this analysis, a
relation was found only between Apo E alleles and
prolactin and LH levels. LH levels were found to be low
in patients with PCOS in the existence of E3 and E4 allele
(p=0.018). However, prolactin levels was statistically lower
in the ApoE4 allele-carrying PCOS patients (p=0.046).
Discussion
In the present study, we compared the frequency of the
ApoE gene polymorphism in PCOS patients and healthy
controls living in Western Turkey. Women with PCOS are
more prone to have metabolic syndrome than healthy
women [12]. Additionally, different markers of atheroscle-
rosis, such as fibrinogen, high sensitive C-reactive protein,
homocysteine, oxidative stress markers, PAI-1, TPA,
carotid intimae-media thickness, and echocardiographic
findings have also been found to be changed [13–16].
Dyslipidemia is very common and includes elevated
triglyceride levels and low high-density lipoprotein (HDL)
cholesterol concentrations in PCOS patients [17].
Dyslipidemia may be the most common metabolic
abnormality in PCOS, although the type and extent of the
findings have been variable. PCOS patients have an
atherogenic lipid profile characterized by lower high-
density lipoprotein (HDL) cholesterol and/or HDL2choles-
terol levels, and higher triglyceride and low-density
lipoprotein (LDL) cholesterol levels than the age- and
Table 1 Clinical characteris-
tics of patients and controls
*mean ± SD
&2-tailed t test
P<0.05 values were accepted
as statistically significant
Characteristic Patients * n=129Control* n=91P&
Age (years)
BMI (k/m2)
Total-cholesterol (mg/dl)
LDL-C (mg/dl)
HDL-C (mg/dl)
Triglycerides (mg/dl)
Fasting Glucose (mg/dl)
Fasting insulin (mIU/ml)
Homocysteine (µmol/L)
FSH (mIU/ml)
LH (mIU/ml)
Estradiol (pg/ml)
DHEA-S (µg/dl)
17-OHP (ng/ml)
Total-Testosterone (ng/ml)
Free-Testosterone (pg/ml)
Prolactine (pmol)
Fibrinogen (mg/dl)
hs-CRP(mg/dl)
24.58±4.61
24.47±4.64
199.86±38.82
117.75±29,35
56.76±13.89
122.34±55.27
91.43±7.55
12.16±9.07
2.63±1,60
5.43±1,77
6.53±3.68
34.90±20.83
225.92±89.32
1.72±0.87
0.99±1.36
3.39±1,80
17.08±7.27
367.00±75.07
0.41±0.63
25.48±3.38
24.2±3.31
187.33±13.77
110.31±11.40
57.33±4.49
115.09±24.07
88.08±3.96
4.71±1.17
1.33±0.22
4.17±1.24
4.18±0.99
39.02±32.35
222.29±42.84
0.90±0.31
1.07±0.69
1,52±0.40
15.51±4.49
277.12±44.07
0.29±0.22
0.115
0.65
0.004
0.023
0.705
0.004
< 0.001
< 0.001
< 0.001
< 0,001
< 0.001
0.254
0.721
< 0.001
0.632
< 0.001
0.07
< 0.001
0.730
J Assist Reprod Genet (2009) 26:1–6 3 3

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weight-matched control women [18]. Thus, the presence of
abdominal obesity, insulin resistance and dyslipidemia
predispose women with PCOS to cardiovascular diseases
(CVDs) [19].
Another study, found that E4 carriers carry a higher risk
of hyperuricaemia and postprandial hypertriglyceridaemia
after a fat excess in patients with metabolic syndrome [35].
Some researchers described a connection between E4 variant
and increased fasting plasma glucose levels and plasma
insulin levels [20, 21]. In our study, E4 allele frequency was
determined to be similar in patient and control groups. Our
results demonstrate a statistically significant increase in
triglyceride, LDL-cholesterol and total cholesterol levels of
the PCOS patients when compared to those of controls.
However, no statistically significant difference was found in
LDL-cholesterol, total cholesterol, triglyceride, and HDL-
cholesterol levels in E3, E4, and E2 alleles of ApoE gene.
Some researchers presented similar study and they demon-
strated similar results in patients with PCOS from Finland
[22]. On the other hand, in Western Anatolia ApoE4 allele
frequency (3.9%) was lower than that of Finland (17.2%) in
women with polycystic ovary syndrome.
Increasing evidence suggests that atherosclerosis is a
chronic inflammatory process and due to this fact the
markers of the inflammatory response such as CRP and
fibrinogen might be useful in assessing the risk of
cardiovascular disease [23]. Women’s Health Study has
shown that CRP is a strong independent risk factor for
cardiovascular diseases in females [24]. Endothelial dys-
function and high concentrations of high-sensitivity CRP
were detected even in normal-weight women with PCOS
[25].
No statistical correlation was found between different
alleles of Apo gene and homocysteine, fibrinogen, and hs-
CRP in our current study. The Asian people usually have
lesser apo ε4 frequency than European people. The APOE4
allele has also been connected to increased plasma LDL-C
concentrations in healthy subjects [26, 27]. The negative
effects of lifestyle factors can interact with genetic factors
(e.g. ApoE4), therefore increasing cardiovascular risk [28,
29]. These values are similar to those in other populations,
such as Italians (ApoE4: 9.4%) and the French (ApoE4:
12.0%) [30, 31]. In our study, the ApoE2 allele frequency
in western Turkish healthy population (16.7%) was found
to be higher than in Caucasian (8.0%) and Greek
populations (8.1%) [32, 33].
Table 3 Biochemical and
Hormonal parameters between
ApoE haplotypes in patient
group
*P<0.05 values were accepted
as statistically significant
Haplotypes and parameters (Mean ± SD)
E2E3 E4P value
Total-cholesterol (mg/dl)
LDL- cholesterol (mg/dl)
HDL- cholesterol (mg/dl)
Triglycerides (mg/dl)
Fasting Glucose (mg/dl)
Basal Insulin (mIU/ml)
Homocysteine(µmol/L)
FSH (mIU/ml)
LH (mIU/ml)
Estradiol (pg/ml)
DHEA-S (µg/dl)
17-OHP (ng/ml)
Total-Testosterone (ng/ml)
Free-Testosterone (pg/ml)
Prolactine(pmol)
Fibrinogen (mg/dl)
188.20±40.13
111.20±29.32
59.00±16.18
110.50±45.34
92.70±6.91
11,20±7..61
2.73±1.62
5.48±1.80
8.50±5.16
38.20±20.70
247.11±127.18
1.98±1.25
0.86±0.29
3.30±1.53
18.36±10.55
343.81±72.34
200.36±39.43
118.12±29.82
55.66±12.37
125.00±54.90
91.03±7.32
12.18±9.56
2.57±1.61
5.54±1.74
6.29±3.33
33.53±19.79
222.39±86.19
1.74±0.85
1.04±1.48
3.48±1.84
17.33±7.09
369.50±74.70
207.40±30.45
121.00±25.76
66.00±22.68
106.90±69.22
94.30±10.37
13.10±5.32
3.13±1.49
4.19±1.80
6.98±5.00
46.00±29.43
240.30±78.69
1.29±0.51
0.63±0.20
2.61±1.52
13.00±3.09
365.70±85.13
0.503
0.712
0.067
0.468
0.360
0.893
0.569
0.069
*0.018
0.167
0.597
0.177
0.503
0.343
*0.046
0.560
Table 2 Distrubution of ApoE haplotypes and genotypes
Haplotypes/
Genotypes
Patients* Control*p&
OR&
95% CI&
E2 11
4.3%
235
91.8%
10
3.9%
11
8.6%
108
83.6%
10
7.8%
30
16.7%
136
75.6%
14
7.8%
31
33.3%
46
51.1%
14
15.6%
< 0.001
R
E3< 0.001
0.2120.103–0.437
E40.513
0.5130.177–1.490
E2/E3 < 0.001
R
E3/E3< 0.001
6.342.93–13.74
E3/E4 0.072
1.940.67–5.66
P<0.05 values were accepted as statistically significant
*% frequency
&Logistic regression analysis
4J Assist Reprod Genet (2009) 26:1–6

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The occurrence of the ApoE4 allele may therefore
contribute to the variation in the risk of these diseases such
as coronary artery disease, hyperlipidemia, type 2 diabetes,
metabolic syndrome, and polycystic ovary syndrome across
populations. ApoE4 allele frequency was not determined to
be higher in patients with PCOS than the controls in our
study.
The ApoE3 allele is the most common allele in each
population [34]. The ApoE3 allele showed a significantly
higher frequency in the PCOS patient group when
compared with the control group (p<0.001). E3/E3 geno-
type of ApoE gene in patients with polycystic ovary
syndrome was found to be statistically significantly higher
than that of healthy control group in this study. But no
relation was found between ApoE gene E3/E3 and lipid
parameters and cardiovascular risk factors such as hs-CRP,
fibrinogen, and homocysteine. However, there is a need for
long-term prospective studies for explaining the relation
between ApoE3 allele elevation and reduction of cardio-
vascular disease risk in patients with PCOS.
Although there was a genotype and allele difference
between control and patient group in this study, no
statistically significant change was determined in lipid and
other cardiovascular risk factors in connection with allele
and genotype. ApoE gene polymorphism has no effect on
cardiovascular risk factors such as lipid and hs-CRP,
fibrinogen, and homocysteine in Turkish patients with
PCOS.
Acknowledgements
assistance in statistical evaluation of this study. We are thankful to
Technicians of Nail Tartaroglu Endocrinology Laboratory for their
assistance in coordinating this study.
We would like to thank Hatice Uluer for her
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