Annals of Human Biology

ISSN: 0301-4460 (Print) 1464-5033 (Online) Journal homepage: http://www.tandfonline.com/loi/iahb20

Is there any association of apolipoprotein E gene

polymorphisms with metabolic syndrome in a
young population of Croatian origin?

Ivana Karmelić, Jasna Lovrić, Tamara Božina, Ana Merkler, Nada Bozina &
Jadranka Sertic

To cite this article: Ivana Karmelić, Jasna Lovrić, Tamara Božina, Ana Merkler, Nada Bozina &
Jadranka Sertic (2016): Is there any association of apolipoprotein E gene polymorphisms with
metabolic syndrome in a young population of Croatian origin?, Annals of Human Biology, DOI:
10.1080/03014460.2016.1210675

To link to this article: http://dx.doi.org/10.1080/03014460.2016.1210675

Accepted author version posted online: 07

Jul 2016.
Published online: 07 Jul 2016.

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 Is there any association of apolipoprotein E gene polymorphisms with metabolic syndrome in

a young population of Croatian origin?

Key terms: obesity, APOE polymorphism, metabolic syndrome, Mediterranean diet, young

population

Dr. Ivana Karmeli* - School of Medicine, Department of Medical Chemistry, Biochemistry

and Clinical Chemistry, Šalata 3, Zagreb, 10000 Croatia

Prof. Jasna Lovri- School of Medicine, Department of Medical Chemistry, Biochemistry and
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Clinical Chemistry, University of Zagreb, Šalata 3, Zagreb, 10000 Croatia

Mrs. Tamara Božina

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- School of Medicine, Department of Medical Chemistry,

Biochemistry and Clinical Chemistry, University of Zagreb, Šalata 3, Zagreb, 10000 Croatia

Ana
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Merkler- School of Medicine, Department of Laboratory Diagnostics, University
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Hospital Centre, University of Zagreb, Kišpati?eva 12, Zagreb, 10000 Croatia

Prof. Nada Bozina- University Hospital Center Zagreb , Clinical Institute for Laboratory
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Diagnosis, Kispaticeva 12, Zagreb, 10 000 Croatia

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- The University of Zagreb, Zagreb School of Medicine, Salata 3,

Zagreb, 10 000 Croatia

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Prof. Jadranka Sertic - Clinical Hospital Center Zagreb, University Zagreb School of
Medicine, Clinical Institute of Laboratory Diagnostics;Department of Medical Chemistry,
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Biochemistry and Clinical Chemistry, University of Zagreb, Kispaticeva 12; Šalata 3, Zagreb,
10000 Croatia

*corresponding author Email: k.ivanakarmelic@gmail.com

 ABSTRACT

Background: Apolipoprotein E has an important role in lipid metabolism and adipocyte

activity and apo E gene (APOE) might serve as a potential determinant of MetS.

Aim: The aim of the presented study was to investigate the association between APOE

polymorphism and MetS in young adult subjects of Croatian origin.

Methods: We measured biochemical and anthropometric parameters of 149 young (aged 20-

33) subjects. The APOE was genotyped by real-time PCR.

Results: No APOE genotype significantly increased the risk for development of MetS.
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Significant association was found between APOE polymorphism and elevated blood pressure

(EBP) (p=0.019). The carriers of the 4 allele had decreased risk for EBP (OR=0.28, 95%CI)

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comparing to 3 allele carriers (3 allele vs. others χ2=7.08; p=0.005). APOE alleles were

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significantly associated with the concentration of TC and LDL-C (χ2=12.11, p=0.002 and
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χ2=15.76, p<0.001, respectively). With diet as modification covariate there was a significant

correlation of APOE alleles with the concentrations of adiponectin and leptin (χ2=7.076;
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p=0.029 and χ2=7.46; p=0.024, respectively).

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Conclusion: Although APOE variants were not confirmed as the risk factor for development

of MetS, the APOE alleles were associated with some of the metabolic parameters in young

Croatian subjects. The relation of APOE alleles with a concentration of adiponectin and leptin
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depends on the diet intake.

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 INTRODUCTION

Metabolic syndrome (MetS) is a combination clustering of factors associated with increased

risk of type 2 diabetes (T2D) and accelerated cardiovascular events (Andreassi, 2009, Grundy

et al., 2014). The core risk factors leading to MetS include increased level of serum

triglycerides (TG), and decrease in the level of high-density lipoprotein-cholesterol (HDL-C),

elevated blood pressure (EBP), glucose (GLUC) intolerance, a pro-thrombotic state and pro-

inflammatory state and each of these ''risk factors'' has several components (Alberti et al.,

2005, Grundy, 2006, Eckel et al., 2010). Although the prevalence of MetS depends on age,
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ethnicity, gender and diagnostic criteria today most investigators agree that rising prevalence

of MetS worldwide is largely due to the increasing incidence of abdominal obesity (Grundy,

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2006, Grundy et al., 2014). Abdominal obesity is associated with quantitative and qualitative

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changes in circulating lipids and lipoproteins as well as with dysregulation in the secretion of
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adipocytokines that participate in the development of the MetS (Matsuzawa, 2006, Karmelic

et al., 2012, Hwang et al., 2015). It is known that pro-inflammatory cytokines (e.g. leptin,
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tumor necrosis factor  and interleukin 6) may promote the obesity-related complications
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which are components of MetS (De Luis et al., 2009). In contrast to other cytokines, the

concentration of anti-inflammatory, circulating adiponectin (ADIPO) was significantly

decreased in obese subjects and in subjects who developed MetS (Matsuzawa, 2006,
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Stojanovic et al., 2015). It is commonly accepted that gene and environment (gender, age, life

style, diet) interactions play a crucial role in determining the risk of complex human diseases
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(Ma and Xu, 2015). Studies in different populations worldwide have described an association

between obesity and MetS and genetic variation in apolipoprotein E gene (APOE) (Huang et

al., 2006, Gao et al., 2007, Kypreos, 2009, Ferreira et al., 2011, Sima et al., 2007, Loos, 2012,

Vucinic et al., 2014, Zarkesh et al., 2012, Luptakova et al., 2013, Zeljko et al., 2011).
 Because of the important role of apoE in lipid and lipoprotein metabolism and adipocyte

activity (Carmel et al., 2009) APOE might serve as a potential determinant of obesity and

MetS. Studies have shown that carriers of 2 allele have lower plasma LDL-cholesterol

(LDL-C) and higher HDL-C than carriers of 3 allele; the opposite finding has been

confirmed for carriers of 4 allele as compared to carriers of 3 (Koch et al., 2008, Gao et al.,

2007, Alvim et al., 2010, Bu, 2009). Endogenous adipocyte APOE expression is an important

modulator of adipocyte lipid metabolisms, influencing TG synthesis, free fatty acid mass,

cholesterol synthesis and gene expression (modulator of adipogenesis) (Huang et al., 2006,
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Huang et al., 2009, Lasrich et al., 2015, Huang et al., 2011). Allele 2 is able to increase the

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expression of genes involved in TG metabolism and oxidation in adipocytes (Huang et al.,

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2006, Ferreira et al., 2011). Published data are quite conflicting most likely due to differences

in age and genetic or ethnic background of the study populations, and environmental
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influence, depending on the degree of obesity of the study population. Gene-diet interactions

could have an important role in modulating phenotypes related to obesity and MetS (Yang et
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al., 2007, Andreassi, 2009). Only a few studies have investigated interactions of APOE and
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environmental factors on development of MetS. Understanding these interactions in the

development of obesity and MetS can significantly help us in understanding the

pathophysiology of these complex diseases and the development of targeted therapies.

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Considering the importance and implications of MetS for healthcare we evaluated whether the
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APOE polymorphism was associated with MetS in the population of young subjects of

Croatian origin. Also, we have analysed whether diet could interfere with APOE

polymorphisms influencing the sensitivity to the MetS.

 METHODS

Study subjects

A total of 149 subjects (65 male, 84 female) aged 20-33 (mean 26±3 years) were chosen

during routine medical check-up (Karmelic et al., 2012). Each participant completed a

questionnaire about family history of the most common chronic disease and risk factors for

this research, such as taking medication that could affect blood pressure, glucose or lipid

metabolism. Also, each participant completed a questionnaire about a type of diet

(Mediterranean-type of diet, continental-type of diet and mixed-type of diet). The

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Mediterranean-type of diet included proportionally high daily consumption of olive oil and

fruits and vegetables, consumption of fish two to three times a week, consumption of white

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meat instead of red meat and consumption of nuts at least three times a week. The continental-

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type of diet included high consumption of red meat and meat products, consumption of small
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quantities of fruits and vegetables and rare consumption of fish without olive oil. The diet that

included elements of both the Mediterranean-type of diet and continental-type of diet is

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categorised as a mixed-type of diet.

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Participants had their blood tests completed and anthropometric measurements taken. All

examinees voluntarily participated and signed informed consent forms, and the study protocol

was approved by the Ethics Committee of the University Hospital Centre Zagreb. MetS was
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defined using criteria established in the International Diabetes Federation (IDF) diagnostic

criteria (Alberti et al., 2005). An individual with a combination of abdominal obesity (defined
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as WC≥94 cm for European men and ≥80 cm for European women) and any two or more of

the following risk factors was classified as having MetS: elevated TG levels (TG≥1.695

mmol/L), reduced HDL-C (HDL-C<1.036 mmol/L for men and <1.295 mmol/L for women),

EBP (systolic blood pressure, SBP≥130 mm Hg or diastolic blood pressure, DBP≥85 mm

Hg), and increased fasting plasma glucose (GLUC≥5.6 mmol/L). Hypercholesterolemia and
 increased LDL-C levels were identified when total cholesterol, (TC)≥5 mmol/L and LDL-

C>3.0 mmol/L were determined, respectively.

Clinical and biochemical parameters determination

The following anthropometric measurements were obtained: body weight (BW), body height

(BH) and WC. BW was measured by standard medical balance. BH was measured using the

stadiometer. BMI was calculated by standard formula BMI=BW/BH (kg)/(m2). WC was

measured twice to the nearest 0.5 and the mean was used for subsequent analyses. Blood
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pressure (BP) measurements were taken in the morning in the sitting position, after 10-15

minutes rest period, through the use of a sphygmomanometer. EBP was defined according to

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IDF criteria by a SBP that was ≥135 mm Hg and a DBP that was ≥85 mm Hg for men and

women, respectively (Alberti et al., 2005). TE

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Blood samples for biochemical analyses (TC, TG, LDL- C, HDL-C, GLUC) were collected

after 12 h overnight fasting and measured by standard laboratory procedures which are
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described in our previous work (Karmelic et al., 2012). Fasting plasma total ADIPO and
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LEPT concentration were determined using a validated sandwich enzyme-linked

immunosorbent assay (ELISA kit, ADIPO: E09 or LEPT E07, Mediagnost Germany). The

values of ADIPO and LEPT were interpreted in relation to BMI, age and sex of subjects.
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APOE genotyping

Total DNA was extracted from human blood samples using standard salting out method
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(Miller et al., 1988). The APOE was genotyped by applying a DNA assay that uses real-time

PCR and fluorescence resonance energy transfer (FRET) with the LightCycler (LC) (Roche

Diagnostics, Mannheim, Germany) (Nauck et al., 2000, Wittwer et al., 1997). In this assay, a

265 bp fragment harbouring exon 4 of the APOE gene is amplified from human genomic

DNA. Analyses for APOE mutations at codons 112 and 158 were performed using primers
 and hybridisation probes commercially available from Roche Diagnostic LightCycler ApoE

Mutation Detection Kit (Roche Diagnostics, Mannheim, Germany). The reaction mixture (20

l) was prepared in a glass capillary for LC using 18 l of the Master mixture and 2 l of the

isolated genomic DNA template or the control template following the manufacturer's

procedure. After initial denaturation at 95°C for 60 sec, 45 PCR cycles were performed with 1

sec of denaturation at 95°C; 10 sec of annealing at 60°C and extending at 72°C for 10 sec.

Fluorescence was measured at the end of the annealing period of each cycle to monitor

amplification. Genotyping was carried out via melting curve analysis. The resulting melting
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peak seen at the different fluorescence channels allowed us to discriminate among the

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homozygous as well as the heterozygous genotypes. Replicate quality control samples were

included and genotyped with 100% concordance.

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Statistical analysis

For descriptive statistical analyses of the obtained data software packages SPSS 17.0 (SSPS
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Inc., Chicago, IL) and MedCalc 10.2.0.0. (Frank Schoonjans, Mariakerke, Belgium) were
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used. One-way Anova test or Kruskal-Wallis test, depending on parametric or nonparametric

distribution, were conducted to explore the impact of diet type on anthropometric and

biochemical parameters. Post-hoc tests indicated among which diet types significant
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differences were present. The comparisons between the groups of subjects with developed

MetS and controls were analysed using Student’s t-test or Mann-Whitney test, depending on
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distribution normality or  test and Fischer exact test for categorical variables. Genotype and

allele frequencies were counted directly. The association of APOE gene variants with the

development of MetS and their components was tested using program UNPHASED ver.

3.0.10. (Dudbridge, 2003). A test for Hardy-Weinberg equilibrium using Markov chain

method (Guo and Thompson, 1992), as implemented in Arlequin ver. 3.01 (Excoffier et al.,
 2005) was applied. The level of significance was set at p<0.05. G*Power 3.1.9.2. Programme

(University of Dusseldorf Germany) was used for statistical power analysis. Within our

chosen sample size and medium effect size, the power (1-) of study was approximately 0.80.

Population sample description

The clinical and metabolic characteristics of participants are shown in Table I. The study

population included 43.6% of men and 56.4% of women (Karmelic et al., 2012). The mean

age was 26.4 years and the median BMI was 24.2 kg/m2 (maximum 44.1 kg/m2). With the
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exception of BMI, ADIPO and LEPT, there were no differences between sexes for the

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variables measured (p>0.05). Therefore, all the subjects were analysed together. 16.1% of

subjects were on Mediterranean-type of diet. There were statistically significant differences

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between diet type in values for WC (p=0.004), BMI (p=0.005) and TG (p=0.031). The
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Mediterranean-type of diet has contributed to lower values of WC, BMI and TG in

comparison with the other two types of diet. However, the results of logistic regression
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confirmed the type of diet only as a predictor of TG concentration (F=8.489, p=0.004).

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Of 149 subjects, MetS was defined in 34 subjects (22.82%) (Table I) (Karmelic et al., 2012).

The results of appropriate tests revealed significant differences (p<0.05) in means or median

of anthropometric and biochemical measurements between two analysed groups for all values
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(Table I.). The variables sex, age and diet were similar between the subjects with or without

MetS. Among the MetS variables (WC, TG, HDL-C, GLUC, SBP and DBP) in all of the
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subjects, increased WC was the most prevalent (46.4% in women and 44.6% in men),

followed by hypertriglyceridemia (21.5%) and hypercholesterolemia (30.9%). Elevated SBP

and DBP were detected in 15.4% and 19.5% of subjects, respectively. Decreased HDL-C was

found in 4.8% of women and 12.3% of men. Increased fasting plasma glucose was detected in

only 4.0% of subjects. MetS variables (DBP, WC, HDL-C and GLUC) and non-MetS
 variables (LDL-C and TC) were significantly different between groups even after adjustment

for BMI (p<0.05). Hyperleptinemia and hypoadiponectinemia were detected in a group of

subjects with MetS. These results are the same as in our previous study, since it is the same

group of patients (Karmelic et al., 2012).

Genetic analysis

The allelic distribution of the 2, 3 and 4 alleles in the APOE gene was 8.4%, 78.9% and

12.1%, respectively. Groups exhibited Hardy-Weinberg equilibrium for allelic distribution

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(p=0.374). The frequencies of the six possible genotypes 2/2, 2/3,2/4, 3/3, 3/4 and

4/4 were 2 (1.3%), 15 (10.1%), 6 (4.0%), 95 (63.8%), 30 (20.1%) and 1 (0.7%),

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respectively. No differences in genotype (p=0.306) and allele (p=0.790) distribution

according to gender were detected.

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Associations of APOE polymorphisms with MetS
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We compared the genotypes and allele distribution of APOE in the group of subjects who met
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met the criteria for MetS (MetS group) and in the group of subjects who did not (control

group). (Table II). The frequencies of the 2, 3 and 4 alleles were not different between the

two groups (p=0.490). The frequencies of APOE genotypes were marginally statistically
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different between the analysed groups (p=0.050) but no genotype significantly increased the

risk for development of MetS. However, the 2/genotype was only present in the group of
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subjects without MetS  genotype vs. others, χ2=35.41, p<0.001).

Associations of APOE polymorphisms with metabolic parameters

Analysis of association of APOE alleles with metabolic parameters is presented in Table III.

APOE alleles were significantly associated with the concentration of TC and LDL-C (p=0.002
 and p<0.001, respectively). APOE genotypes 2/2 and 2/3 were associated with lower

concentrations of TC (p<0.001 and p=0.029, respectively) and LDL-C (p<0.001 and p=0.002,

respectively (data not shown in the Table III). We introduced type of diet as an environmental

factor in the analysis of the association of APOE alleles with metabolic parameters (Table

IV). Besides the existing relationship of APOE alleles with concentrations of TC and LDL-C,

with diet as a modification covariate, there was a significant correlation with the

concentrations of ADIPO and LEPT (p=0.029 and p=0.024, respectively).

Associations of APOE polymorphisms with elevated blood pressure

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We compared the APOE genotype distribution between the group of subjects with EBP (SBP

≥130 mmHg and/or DBP ≥85 mmHg) and the group of subjects who did not met the criteria

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for EBP (SBP <130 mmHg and/or DBP < 85mmHg) to examine if the APOE polymorphism

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was significantly related to EBP (Table V). The genotype distribution of APOE was different
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between subjects with and without EBP (p=0.019). In relation to EBP, a significant

association was found for 3/3 variant (84.6 vs. 56.3 %). Among the subjects with EBP, the
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frequencies of 2/3 and 3/4 genotypes were significantly lower compared to the subjects
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without EBP (2.6 vs. 12.7% and 7.7 vs. 24.6%, respectively). Only the subjects with 3/4

genotype had slightly decreased risk for development of EBP (OR 0.21, [95% CI 0.06-0.74],

p=0.015) than 3/3 genotype carriers (3/3 genotype vs. others χ2 =12.5; p<0.001). The
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frequencies of the 2, 3 and 4 alleles were significantly different between the two groups
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(p=0.012). The 4 allele was more frequent in the individuals without EBP (15.4 vs. 5.1%;

p=0.002) and the carriers of the 4 allele had decreased risk for the development of EBP (OR

0.28 [95% CI 0.09-0.81], p=0.019) than 3 allele carries (3 allele vs. others χ2=7.08;

p=0.005).
 DISCUSSION

In this work we tested the role of APOE polymorphism as a marker of MetS among young

adults of Croatian (European-derived) origin. Metabolic syndrome is largely unrecognised,

especially in young overweight subjects. The age stratified prevalence of overweight and

obesity in Croatia showed the largest increase in the youngest age group of subjects (18-34

years). Therefore, the youngest adults were chosen as a priority group of the population in

order to reduce the obesity epidemic as well as MetS. (Karmelic et al., 2012, Kolcic et al.,

2010). The prevalence of MetS in the population differs depending on diagnostic criteria, age,
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ethnicity, sex and geographical area and usually varies between 22 and 39% (Khunti and

Davies, 2005, Thaman, 2013). According to IDF diagnostic criteria MetS was defined in

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22.82% (29.2% of males and 17.8% of females) of participants (Karmelic et al., 2012) which

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is a rather large proportion for such a young group of participants. The aetiology of MetS is
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complex and includes environmental and genetic factors as well as their interactions (Kaur,

2014). In our study, subjects who consumed the Mediterranean-type of diet had the lowest
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values of anthropometric parameters as well as the lower TG levels compared to the other two
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types of diet. This confirms the assumption that type of diet can have a significant impact on

people's health (Sertic et al., 2009, Babio et al., 2009, Tortosa et al., 2007, Phillips, 2013).

Indeed, data suggest that diets higher in unsaturated fatty acids, particularly monounsaturated
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fatty acids like the Mediterranean diet have several advantages over the other types of diets

(e.g. continental-type of diet) (Gotsis et al., 2015, Camargo et al., 2014).

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In our study 50.75% of participants with abdominal obesity have MetS, suggesting that

progression to MetS is highly dependent on genetic background (Karmelic et al., 2012, Povel

et al., 2011).

APOE is polymorphic in the entire human population, but with a different allele frequency,

which largely depends on the studied population. In our study the 3 allele is the most
 prevalent, followed by the 4 and 2 alleles. This distribution was also observed in other

European-derived populations (Ferreira et al., 2011, Eisenberg et al., 2010). In recent years,

the link between APOE polymorphisms and obesity as well as MetS has been studied, but the

results are quite contradictory. Since ethnicity and environmental factors have been reported

to be a major determinant of genetic variability that could affect the outcome of the study

(Grundy et al., 2014).

In our study no significant association was found between APOE genotypes/alleles and

increased risk of MetS. In contrast to our research an epidemiological study among the Roma
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minority population of Croatia has shown the presence of increased frequency of 4 in

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patients with MetS (Zeljko et al., 2011). Similarly, a study of older women showed the

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association of the phenotypes of apo-E4/4 and apo-E3/4 with increased WC (Sima et al.,

2007). Research on extreme obese subjects showed association of 4 allele with obesity and
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MetS (Ferreira et al., 2011). However, research among predominantly white US men and

women does not support an association between APOE polymorphism and MetS (Lai et al.,
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2015).
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Numerous studies revealed the influence of APOE genotypes on lipid and lipoprotein levels.

Predominantly it was shown that 2 allele is associated with reduced levels of LDL-C and TC

while the opposite finding has been confirmed for 4 allele carriers (Alvim et al., 2010). In
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this study, we confirmed that 2 allele contributes to reducing the concentration of TC as well
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as the reduction in LDL-C level. We did not find a significant association between APOE

alleles and plasma concentration of TG as well as between APOE alleles and plasma

concentration of ADIPO and LEPT. In contrast, some studies demonstrate that the 2 allele

was associated with hypertriglyceridemia, i.e. increased serum concentrations of TG in 2

homozygous and heterozygous allele carriers in relation to most common 3/3 genotypes

carriers (Murdoch et al., 2007, Dallongeville et al., 1992). A statistically significant

 association between saturated fat intake with increased VLDL cholesterol, decreased HDL-C

and smaller LDL particles in APO 2 carriers has been reported (Talmud, 2007, Gotsis et al.,

2015, Camargo et al., 2014). This suggests that intake of diet, which is high in saturated fatty

acids (such as continental-type of diet) may result in elevated plasma concentration of LDL-C

and TC in 2 carriers. This confirms the claim that dietary habits cause metabolic response

that could show a relative sensitivity for carriers of different variants of genes for apoE

(Balwierz et al., 2009, Das and Puskas, 2009, Zeljko et al., 2011). The importance of the

gene-environment interaction may play a role in the early development of disease only when
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people with high risk genetic profiles enter a high risk environment (Andreassi, 2009, Zeljko

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et al., 2011, Phillips, 2013). At the population level, people with high risk for MetS may

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benefit from early intervention implemented in the diet. Sertic et al have evaluated that a

Mediterranean-type of diet, high in unsaturated fatty acids, decreases TG concentration and

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this effect is dependent on APOE genotypes (Sertic et al., 2009).

It is also shown that the Mediterranean-type of diet increases LDL-particle size compared to
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continental-type of diet and this effect is dependent on APOE genotypes (Moreno et al.,
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2004). Therefore, it is possible that gene-diet interactions are responsible for the observed

association between 2 allele and obesity in previous research (Talmud, 2007, Boer et al.,

1997). While the association of APOE polymorphisms with lipid status is not a new finding,
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in our study we found the association of APOE polymorphisms with ADIPO and LEPT
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concentrations in young adults of Croatian (European-derived) origin. When we introduced

diet as a modification variable, the association of APOE variants with the serum concentration

of ADIPO and LEPT have become significant. We may assume that the endogenous apoE in

adipocytes, through modulation of lipid metabolism, affects the concentration of those

adipokines and this effect isdependent on diet type and APOE genotype. In our previous

study, we confirmed the protective /positive impact of the ADIPO on the MetS components
 (Karmelic et al., 2012). We demonstrated significant inverse correlations between ADIPO

plasma concentration and TG in this population.

The role of LEPT in pathogenesis of obesity can be inferred by measurement of plasma leptin.

An increase in plasma LEPT suggests that obesity is the result of resistance of leptin while a

low concentration of leptin in the context of obesity suggests decreased production of leptin

(DePaoli, 2014). A set of in vitro experiments using cultures of primary preadipocytes and

adipocytes have shown a possible role of APOE in adipogenesis (Chiba et al., 2003, Huang et

al., 2009) and on APOE expression from adipocytes (Kypreos et al., 2009). The results of
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experiments have shown that the APOE expression in mature adipocytes depends on the way

the mice were fed and diet-induced obesity was associated with the reduced expression of

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APOE in adipocytes (Huang et al., 2007).

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The relationship between APOE variants and hypertension is not well established, and results
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of studies are contradictory due to differences in age and ethnic background of study

populations (Li et al., 2003, Wei et al., 2015). Our study is consistent with another study
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which indicates that the 4 allele was related to lower blood pressure in overweight and obese
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subjects (Ferreira et al., 2011, Cho et al., 2009).

Many conflicting data found in the literature on the associations between MetS and individual

gene influences could be due to different gene-gene interactions. Since many genes are
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involved in the regulation of MetS phenotypes, or phenotypes of some of its traits, in future

studies we suggest studying not only gene-environment, but also gene-gene interactions.
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CONCLUSION

In conclusion, APOE polymorphisms do not affect the risk of MetS probably due to the low

number of subjects included in the study. The 2 allele carriers have significantly decreased

concentrations of TC and LDL-C while 4 allele was associated with lower blood pressure as
 compared to other alleles and this is not a novel concept. The established relation of APOE

alleles with a concentration of LEPT and ADIPO, which depends on the diet intake, is a

remarkable result of the present study.

Study Limitations

There are several limitations in this study. A rather low number of subjects were included.

But, data from this study could be used to design a larger confirmatory study which is needed

to confirm the associations obtained in this research. We did not evaluate the effects of other

environmental factors (physical activity, smoking) except diet in this study. The effects of diet
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and other environmental factors should be examined in more detail in future. For these

reasons, the findings from this study cannot be directly generalised to the Croatian population

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and further larger studies are required to confirm these results.

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ACKNOWLEDGEMENTS

Declaration of interest: The authors report no conflicts of interest. The authors alone are
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responsible for the content and writing of the paper.

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 Table I. Clinical and metabolic features of study participants

Parameter All subjects Without MetS With MetS p

n 149 115 34

Age (years) 26.4 (20-35) 25 (20-35) 30.5 (20-35) <0.001

SEX ♂65 ♀84 ♂46 ♀ 69 ♂19 ♀ 15 0.118

DIET Mediterranean 24 (16.1 %) 21 (87.5 %) 4 (12.5 %)

Continental 105 (70.5 %) 80 (76.2%) 25 (23.8%) 0.322

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Mixed 20 (13.4 %) 14 (70.0%) 6 (30.0%)

BMI (kg m-2) 24.2 (19.9-44.1) 23.0 (19.9-42.8) 30.3 (25.0-44.1) <0.001

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WC (cm) 88.1 ± 15.6 82.4 ± 11.5 107.3 ± 11.9 <0.001

TG (mmol/L) 1.1 (0.34-5.7) TE

0.95 (0.34-4.2) 1.9 (0.95-5.74) <0.001
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TC (mmol/L) 4.7 ± 1.1 4.5 ± 0.96 5.1 ± 1.3 0.008

HDL-C (mmol/L) 1.4 ± 0.3 1.5 ± 0.3 1.1 ± 0.2 <0.001

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LDL-C (mmol/L) 2.7 (1.0-6.30) 2.4 (1.0-6.3) 3.0 (1.1-5.7) 0.004

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GLUC (mmol/L) 5.1 (3.5-8.3) 5.0 (3.5-6.6) 5.3 (4.3-8.3) <0.001

DBP (mmHg) 89.7 ± 10.8 80.0 ± 8.3 87.5 ± 14.3 <0.001

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SBP (mmHg) 120 (90-180) 120 (90 -145) 138.5 (107-180) <0.001

ADIPO (mg/L) 9.0 (0.87-38.5) 12.36 (1.0-38.5) 4.77 (0.87-8.3) <0.001

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LEPT (mg/L) 10.5 (5.4 - 10.8) 6.3 (0.08-58.9) 12.3 (0.29-31.4) 0.029

These results (except concentration of leptin) are the same as in our previous study, since it is

the same group of patients (Karmelic et al, 2012).

BMI, body mass index; WC, waist circumference; TG, triglycerides, TC, total cholesterol;

HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol;

 GLUC, glucose; DBP, diastolic blood pressure; SBP, systolic blood pressure; ADIPO,

adiponectin; LEPT, leptin.

Data are expressed as mean ± standard deviation (SD) or median (range) depending on

parametric or nonparametric distribution. Differences between groups were evaluated by

Student’s t-test or Mann-Whitney test, depending on distribution normality.

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 Table II. Distribution of the APOE genotypes and alleles in the group of subjects with and

without MetS (control)

*
Genotype/allele Control MetS OR (95%CI), p Specific p p

(n=115) (n=34)

n (%) n (%)

3/3 69 (60.0) 26 1 (1-1) χ2=3.70, p=0.054

(76.5) χ2=11,1;

2/3 15 (13.1) 0 (0) 0.08 (0.005-1.46), χ2=35.41, df=5;

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0.090 p<0.001 p=0.050

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3/4 25 (21.7) 5 (14.7) 0.53 (0.18-1.53), 0.242 χ2=0.97, p=0.325

2/2 1 (0.8) 1 (2.9)

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2.65 (0.16-44.01),

0.496
χ2=0.59, p=0.439
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2/4 4 (3.5) 2 (5.9) 1.32 (0.23-7.68), 0.752 χ2=0.31, p=0.576

4/4 1 (0.9) 0 (0) 0.87 (0.03-22.14),

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0.935
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3 178 57 1 (1-1) χ2=1.11, p=0.292 χ2=1.43;

2 (77.4) (83.8) 0.59 (0.22-1.56), 0.359 χ2=0.98, p=0.410 df=2;

4 21 (9.1) 4 (5.9) 0.71 (0.28-1.80), 0.433 χ2=0.62, p=0.432 p=0.490

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31 (13.5) 7
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(10.29)

MetS, metabolic syndrome; OR, odds ratio; Cl, confidence interval

*Specific p = individual vs. other genotypes

Comparisons of subject groups were performed using χ2 test.

 Table III. Association of APOE alleles with the metabolic parameters

Metabolic parameter

Allel TG TC LDL-C HDL-C GLUC ADIPO LEPT

e (mmol/L (mmol/L (mmol/L (mmol/L (mmol/L (mg/L) (mg/L)

) ) ) ) )

2 Spec p=0.425 p=0.032 p=0.002 p=0.880 p=0.556 p=0.091 p=0.966

3 .p p=0.382 p=0.798 p=0.655 p=0.984 p=0.686 p=0.280 p=0.790

4 p=0.066 p=0.022 p=0.051 p=0.919 p=0.956 p=0.919 p=0.845

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p χ2=3.69; χ2=12.11 χ2=15.76 χ2=1.31; χ2=0.33; χ2=4.31 χ2=0.09

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df=2; ; ; df=2 df= 2 ; ;

p=0.158 df=2

p=0.002
df=2

p<0.001
p=0.520
TE p=0.848 df=2 df=2

p=0.116 p=0.952
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TG, triglycerides, TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C,

low-density lipoprotein cholesterol; GLUC, glucose; ADIPO, adiponectin; LEPT, leptin

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Spec. p, Specific p = individual vs. other alleles

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 Table IV. Association of APOE alleles with the metabolic parameters including diet as

modifier covariate

Metabolic parameter

Allel TG TC LDL-C HDL-C GLUC ADIPO LEPT

e
(mmol/L (mmol/L (mmol/L (mmol/L (mmol/L (mg/L) (mg/L)

) ) ) ) )

2 Specifi p=0.880 p=0.038 p=0.007 p=0.647 p=0.548 p=0.685 p=0.98

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3 p=0.984 p=0.159 p=0.018 p=0.379 p=0.259 p=0.068

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p=0.00
4 p=0.919 p=0.998 p=0.970 p=0.151 p=0.258 p=0.008

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p=0.00

2
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p χ2=0.024 χ2=15.76 χ2=10.35 χ2=1.80; χ2=1.443 χ2=7.076 χ2=7.46

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; df= 2 ; ; ; ; ;
df=2

p=0.988 df= 2 df=2 df=2 df=2 df=2

p=0.406
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p=0.039 p=0.006 p=0.485 p=0.029 p=0.02

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TG, triglycerides, TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C,

low-density lipoprotein cholesterol; GLUC, glucose; ADIPO, adiponectin; LEPT, leptin

Spec. p, Specific p = individual vs. other alleles

 Table V. Distribution of the APOE genotypes and alleles in the group of subjects without EBP

– control (SBP<130 mmHg and DBP<85 mm HG; control) and with EBP (SBP≥130 mmHg

and DBP≥85 mm HG; EBP)

*
Genotype/ Control EBP OR (95%CI), p Specific p p

allele (n=110) (n=39)

n (%) n (%)

3/3 62 (56.3) 33 1 (1-1). χ2=12.5, p<0.001

2/3 14 (12.7) (84.6) 0.13 (0.02-1.07), 0.058 χ2=7.99, p=0.005 χ2=13.58;

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3/4 27 (24.6) 1 (2.6) 0.21 (0.06-0.74), 0.015 χ2=8.28, p=0.004 df=2;

1.89 (0.11-31.01), 0.659 χ2=0.46, p= 0.497

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1 (0.9) 3 (7.7) p=0.019
2/2
χ2=0.40, p=0.527
2/4

4/4
5 (4.5)

1 (0.9)
1 (2.6)

1 (2.6) TE
0.38 (0.04-3.35), 0.381
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3 165 (75.0) 70 1 (1-1) χ2=7.08, p=0.005 χ2=8.814;

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2 21 (9.5) (89.7) 0.45 (0.15-1.35), 0.156 χ2=1.48, p=0.224 df=1;

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4 34 (15.4) 4 (5.1) 0.28 (0.09-0.81), 0.019 χ2=9.34, p=0.002 p=0.012

4 (5.1)

SBP, systolic blood pressure, DBP, diastolic blood pressure; EBP, elevated blood pressure;
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OR, odds ratio; Cl, confidence interval

*Specific p = individual vs. other genotypes

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Comparisons of subject groups were performed using χ2 test.