Gene 549 (2014) 209–213

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Apolipoprotein E genotyping in women with recurrent pregnancy loss:An in silico and experimental hybrid study

Ahmad Poursadegh Zonouzi a, Davoud Farajzadeh a,b,⁎, Nasrin Bargahi b,1, Malak Farajzadeh b,1

a Department of Cellular and Molecular Biology, Biological Science Faculty, Azarbaijan Shahid Madani University, Tabriz, Iranb Department of Genetic, Biotechnology Research Center, Research & Development Complex, Tabriz University of Medical Sciences, Tabriz, Iran

Abbreviations: RPL, recurrent pregnancy loss; ApoPolymorphism Phenotyping; OR, odds ratio; CI, confidenc⁎ Corresponding author at: Department of Genetic, B

Research & Development Complex, Tabriz University ofDepartment of Cellular and Molecular Biology, BiologicShahid Madani University, Tabriz, Iran.

E-mail address: farajzadeh@azaruniv.ac.ir (D. Farajzad1 Equal contribution.

http://dx.doi.org/10.1016/j.gene.2014.07.0550378-1119/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 April 2014Received in revised form 2 July 2014Accepted 21 July 2014Available online 23 July 2014

Keywords:Apolipoprotein EApo E ε4 carriersRecurrent pregnancy lossPolyPhen-2

The role of apolipoprotein E gene polymorphisms in the pathogenesis of recurrent pregnancy loss remains con-troversial. Therefore, our objective was to investigate the association between recurrent pregnancy loss and apo-lipoprotein E gene polymorphisms among northwest Iranian women, and also to predict the impact of thesenonsynonymous single nucleotide polymorphisms on structure and function of apolipoprotein E protein. Thesubjects of our current study consisted of 100women that have had two or more consecutive idiopathic first tri-mester miscarriages, and one hundred healthy women from the same geographical areas were used as a controlgroup. After DNA extraction, we used a polymerase chain reaction–restriction fragment length polymorphism togenotype of the apolipoprotein E gene. In addition, we predicted the possible effects of amino acid substitutionsat codons 112 and/or 158 on the structure and function of apolipoprotein E protein using Polymorphism Pheno-typing online software v2. Our results showed that the rate of apolipoprotein E ε4 carriers and the frequency ofthe ε4 allele in the case groupwere statistically and significantly higher than those in the control group (P b 0.05).Therefore, our data support the association of the Apo ε4 allele with RPL; however, in silico analysis predictedthat the amino acid substitution at residue 112 (Apo ε4 allele) is a benignmutation. Accordingly, further studiesare required to elucidate themechanism(s) underlying the link between RPL pathogenesis and the Apo ε4 allele.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Recurrent pregnancy loss (RPL) is a very common obstetric complica-tion and affects between 1 and 5% of reproductive-aged women (Pandeyet al., 2005). The precise pathophysiological mechanism of RPL has notbeen elucidated upon despite a great amount of researches undertakenin this field. It is widely accepted that RPL is a multi-factorial disorderand various factors have been identified, including chromosomal ab-normalities, uterine anatomic malformation, endocrine dysfunction,maternal inherited thrombophilias, infections, immunological, andenvironmental factors (Ford and Schust, 2009; Garcia-Enguidanoset al., 2002; Meka and Reddy, 2006; Zolghadri et al., 2011). However,the exact underlying etiologies have remained inexplicable in about50% of cases (Allison and Schust, 2009).

E, apolipoprotein E; PolyPhen,e interval.iotechnology Research Center,Medical Sciences, Tabriz, Iran;al Science Faculty, Azarbaijan

eh).

Althoughmany studies in recent years have focused on the genet-ic polymorphisms in coagulation cascade, fibrinolysis, thrombophilic(Coulam et al., 2006; Goodman et al., 2006; Yenicesu et al., 2010), cyto-kines (Daher et al., 2003), histocompatibility antigens (Abbas et al.,2004) and metabolic enzymes (Nair et al., 2012) to find out candidategenes for RPL, the genes involved in RPL are still an unanswered enigma.Therefore, it is necessary to investigate the role of new candidate genesto disclose the etiology of RPL. One candidate gene is apolipoprotein E(Apo E), which plays a key role in themetabolism of cholesterol and tri-glycerides (Mahley, 1988). The Apo E gene is located on the long arm ofchromosome 19q13.2 and has three allelic variants ε2, ε3, and ε4encoding three isoforms of Apo E2, E3, and E4, respectively (Das et al.,1985). The differences of the allelic variants are located at exon 4 andcodon positions 112 and 158. E2 allele has cysteine at both positions(Cys 112, Cys 158), E4 allele has arginine at both positions (i.e., Arg112 and Arg 158) and E3 allele has cysteine at 112 and arginine at158 positions (Cys 112, Arg 158). A combination of these alleles gener-ates E2/E2, E3/E3, and E4/E4 homozygous and E2/E3, E2/E4 and E3/E4heterozygous genotypes in human populations (Walden and Hegele,1994).

Of the three distinct alleles (ε2, ε3, and ε4), ε3 is the most commonallelic variant and the frequency of ε3 is nearly 75% among most popu-lations (Walden andHegele, 1994).While ε2 allele is associatedwith an

Table 1The genotypic and allelic frequencies of Apo E gene polymorphisms in case and controlgroups.

Case group,n (%)

Control group,n (%)

P valuea Oddsratios

95% confidenceinterval

GenotypesE2/E2 0 (0.0) 0 (0.0) ND ND NDE2/E3 7 (7) 4 (4) 0.537 1.806 0.512–6.376E2/E4 6 (6) 0 (0.0) 0.029 0.940 0.895–0.988E3/E3 64 (64) 96 (96) 0.000 0.074 0.025–0.218E3/E4 21 (21) 0 (0.0) 0.000 0.790 0.714–0.874E4/E4 2 (2) 0 (0.0) 0.497 0.980 0.953–1.008

Apo E allele frequencyε2 13 (6.5) 4 (2) 0.279 3.128 0.616–15.886ε3 156 (78) 196 (98) 0.000 0.072 0.017–0.317ε4 31 (15.5) 0 (0.0) 0.000 0.840 0.771–0.915

ND: not determined.a P values were calculated by Fisher's exact tests.

210 A. Poursadegh Zonouzi et al. / Gene 549 (2014) 209–213

increased risk for cardiovascular disease and hyperlipoproteinemia (Etoet al., 2002), ε4 allele is considered as a risk factor for Alzheimer's dis-ease (AD), diabetes and ischemic heart disease (Haan et al., 2003;Messier, 2003). The ε4 allele is also associated with inflammatory re-sponses, platelet function, apoptosis and modulation of oxidative stress(Korkmazer et al., 2013).

Recently, many studies have been conducted to discover the link be-tween Apo E polymorphisms and RPL but these studies have shownconflicting results (Agarwal et al., 2010; Asgari et al., 2013; Goodmanet al., 2009; Korkmazer et al., 2013; Ozornek et al., 2010). Therefore,the potential role of Apo E polymorphisms in RPL pathogenesis hasbeen controversial. In the current study, our aim is to address the questionof “whether Apo E polymorphisms can be a risk factor for RPL or not,” byexamining Apo E polymorphisms in women with RPL. In addition, weused Polymorphism Phenotyping online software v2 (PolyPhen-2) to dis-cover the effect of two different residue changes on the function andstructural properties of the Apo E protein.

2. Material and methods

2.1. Subjects or preparation of samples

The investigation was carried out on one hundred women with ahistory of at least two first-trimester idiopathic recurrent miscarriages,who were admitted to Women's Reproductive Health Research Center,Tabriz, Iran, during 2011–2013. Additionally, one hundred healthywomen with at least two successful pregnancies and nomiscarriageswere considered as the control group. For this purpose, the patients withthe following symptoms were excluded from the case group: chromo-somal abnormality, uterine anomalies, genital infections, endocrinologicaldisturbances in luteinizing hormone (LH), follicle-stimulating hormone(FSH) and thyroid stimulating hormone (TSH), anti-phospholipid anti-bodies, and antinuclear antibodies (ANAs). The present study was ap-proved by the Ethics and Human Rights Committee of Tabriz Universityof Medical Sciences and informed consents were filled by all participants.

2.2. DNA extraction and genotype screening

Peripheral blood samples (5 ml from each) were taken from all par-ticipants and their genomic DNA was extracted from the whole bloodusing the salting-out method as described by Miller et al. (1988). ApoE genotype analysis was performed by the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique. Toamplify the desired DNA fragments, the primers ApoF (5′-AGACGCGGGCACGGCTGTCCAAGGA-3′) and ApoR (5′-CCCTCGCGGGCCCCGGCCTGGTACAC-3′) (Agarwal et al., 2010; Procopciuc et al., 2011) were usedin the PCR procedure. The PCR solutions contained 100 ng genomicDNA, 1× PCR buffer, 10 pmol of each primers, 10 nmol of each deoxyri-bonucleotide triphosphates, 1.5mmolMgCl2 and 1 U Taq polymerase inafinal volumeof 25 μl. Initial denaturation at 94 °C for 5minwas follow-ed by 35 cycles of denaturation at 95 °C for 50 s, annealing at 60 °C, ex-tension at 72 °C for 40 s and final extension was performed at 72 °C for7 min. The PCR products were separated on 2% agarose gel and visual-ized with ethidium bromide staining. Finally, the amplified productswere digested by Hha-I restriction enzyme and the digested fragmentswere separated by electrophoresis procedure on 8% polyacrylamidegels stained by silver nitrate. The ε2, ε3, and ε4 alleles were character-ized by the presence of the (91 and 83 bp), (91, 48 and 35 bp) and(72, 48, 35 and 19 bp) fragments respectively (Agarwal et al., 2010).

2.3. Statistical analysis

Statistical analysis was performed using SPSS software version 16.0(SPSS Inc., Chicago, IL, USA). The chi-square (χ2) and Fisher's exacttests were used for calculation of genotype and allele frequencies. TheE2/E4, E3/E4 and E4/E4 genotypes of each group were unified as ε4

carriers and then the values of odds ratios and 95% of confidence inter-vals (CI) were calculated. P values less than 0.05 were statistically con-sidered as significant.

2.4. In silico analysis of nonsynonymous SNPs in Apo E gene

Predicting the putative effects of amino acid substitutions at codonpositions 112 and 158 on the structure and function of Apo E proteinwas performed using PolyPhen-2 online software (http://genetics.bwh.harvard.edu/pph2/index.shtml) (Adzhubei et al., 2010).

3. Results

The mean age of the subjects in the case and control groups was31.96 ± 4.76 years (ranged between 21 and 40), and 29.64 ±3.63 years (ranged between 22 and 38), respectively. Themean numberof miscarriages in the case group was 2.93 ± 1.15 (averaged between 2and 6), and the mean number of successful pregnancies in the controlgroup was 2.38 ± 0.77 (averaged between 2 and 5).

Genotype distribution and allele frequencies in both groups aredisplayed in Table 1. The prevalence of E3/E4 genotype in patients wassignificantly higher than that in the healthy control (P b 0.0001). Inthe control group 96 individuals featured the E3/E3 genotype, whereas64 individuals from the case group had such genotype. This difference isstatistically significant (P b 0.0001). We also observed no E2/E2 geno-type among all the studied participants. Regarding the distribution ofE2/E3, E2/E4 and E4/E4 genotypes, we detected no statistical differencesbetween the case and control groups (P N 0.05). However, the frequen-cies of these genotypes were higher in the case group compared withthe control (see Table 1).

The frequencies of ε2, ε3, and ε4 alleles for Apo E genewere also cal-culated in both case and control subjects (see Table 1). Our results re-vealed that the ε4 allelic frequency was 15.5% in case group, whereasnone of the 100 healthy women carried the ε4 allele. Therefore, thisdifference was statistically significant (P b 0.0001). The ε2 allelic fre-quency in patients suffering from RPL was higher than that in healthywomen, but did not show a statistically significant difference (P N 0.05).

We also unified the E2/E4, E3/E4 and E4/E4 genotypes of the twogroups into a new group (ε4 carrier group), where the odds ratios and95% confidence intervals were calculated. When the E2/E4, E3/E4 andE4/E4 genotypes of the two groups were unified into a new group (ε4carrier group), and the odds ratios and 95% confidence intervals werecalculated, resulted to a strong positive association between carryingε4 allele and RPL (see Table 2).

The substitution of arginine by cysteine at codon 158 (ε2 allele) waspredicted to be deleterious by the use of PolyPhen-2.Whereas, replace-ment of cysteine to arginine at codon 112 (ε4 allele) has no significanteffect on protein function (see Fig. 1).

Table 2Odds ratios (95% confidence intervals) for Apo E gene polymorphisms in case and control groups.

Case group (n) Control group (n) P valuea Odds ratios 95% confidence intervals

ε2 carrier (E2/E2, E2/E3 and E2/E4) 13 4 0.040 3.58 1.12–11.41ε3 carrier (E2/E3, E3/E3 and E3/E4) 92 100 0.007 0.92 0.868–0.975ε4 carrier (E2/E4, E3/E4 and E4/E4) 29 0 0.000 0.29 0.213–0.394

a P values were calculated by Fisher's exact tests.

211A. Poursadegh Zonouzi et al. / Gene 549 (2014) 209–213

4. Discussion

Apo E – a glycoprotein containing 299 amino acids – is a structuralcomponent of chylomicrons, very low density lipoprotein (VLDL),intermediate-density lipoprotein (IDL), and high-density lipoprotein(HDL), which plays a crucial role in lipoprotein and cholesterol metabo-lism and transport by binding to LDL receptor (Mahley, 1988). Apo E hastwo structural domains: the N-terminal domain (amino acids 1–191)contains the binding site for the LDL receptor, while the C-terminal do-main (amino acids 225–299) contains the major lipid binding region(Mahley and Rall, 2000).

The genetic polymorphisms of Apo E affect lipoprotein and cholesterolmetabolism and transport. Individuals carrying the ε2 allele have lowerlevels of plasma cholesterol and elevated plasma levels of triglyceride

Fig. 1. The analysis of the studied Apo E polymorphisms using PolyPhen-2. A) The substitutioreplacement of cysteine by arginine at codon 112 (ε4 allele) has no significant effect on the pr

(TG) compared with individuals carrying the ε3 allele, whereas individ-uals with the ε4 allele display higher levels of plasma cholesterol, espe-cially LDL (Song et al., 2004). An elevated level of plasma lipids leads tothe formation of venous thrombosis through accumulation in the intimaeof blood vessels and activation of inflammatory responses in epithelialcells started by cytokines (Asgari et al., 2013). As pregnancy is a hyper-coagulable state, pregnant women are susceptible to blood clotting.Therefore, it is not surprising that the presence of ε4 allele bolstersthis process due to the lower affinity of E4 containing lipoproteins forLDL receptors that elevate the plasma concentration of cholesterol(Goodman et al., 2009). The formation of thrombus in placenta vesselsdecreases placental blood flow and oxygen delivery to the fetus, leadingto the death of the trophoblast cells, and consequently, fetal death(Jeddi-Tehrani et al., 2011). These detrimental events can be triggered

n of arginine by cysteine at codon 158 (ε2 allele) was predicted to be deleterious. B) Theotein function.

212 A. Poursadegh Zonouzi et al. / Gene 549 (2014) 209–213

by inflammatory cytokines during pregnancy. The Apo E ε4 allele is as-sociatedwith increased levels of the inflammatory cytokines TNF-α andinterleukin 6 (IL6) in animals (Goodman et al., 2009). Thus, the balancebetween inflammatory and anti-inflammatory cytokines is necessaryfor a successful pregnancy. Among the anti-inflammatory cytokines,IL10 seems to be the most important molecule which inhibits the activ-ity of inflammatory cytokines (Zhang et al., 2011).

IL10mediatesmany anti-inflammatory pathways that are critical forsuccessful implantation and formaintenance of pregnancy. However, inApo E4 carriers, the plasma level of this cytokine is effectively reducedand anti-inflammatory activity is attenuated (Asgari et al., 2013). Onthe other hand, the presence of the ε4 allele reduces the affinity ofApo E for LDL receptors which leads to decreased absorption of choles-terol from the bloodstream (Ozornek et al., 2010). It should be notedthat cholesterol is a precursor for the biosynthesis of steroid hormones,which plays an important role in the maintenance of pregnancy (Fryeet al., 2011). Therefore, the Apo ε4 allele can lead to disturbance in pro-duction of steroid hormones, and subsequent fetal loss. These findingspropose that the presence of the Apo ε4 allele led to RPL with variousmolecular mechanisms, including: formation of thrombus in placentavessels, disturbance in the balance between inflammatory and anti-inflammatory cytokines, and disturbance in biosynthesis of steroid hor-mones. However, further studies may be required to find other possiblemechanisms of the Apo ε4 allele in pathogenesis of RPL.

In the current study, we investigated the possible association be-tween Apo E gene polymorphisms and RPL. Our results showed thatapolipoprotein E ε4 carriers and ε4 allele frequencies in patients weresignificantly higher statistically than in the healthy controls in our stud-ied population (P b 0.05) (see Tables 1, 2). Therefore, it appears thatthere may be a positive association between Apo E polymorphismsand RPL, and the presence of the Apo E ε4 allele increases risk of earlyfetal loss. Our results are in accordance with the findings of several pre-vious studies that have been published in thisfield. Asgari et al. reportedthe positive association of Apo E polymorphism with RPL in a case–control study. Though, they reported similar data from Tehran, Iran, thepopulation they had studied was ethnically different from the NorthwestIranian population, the Azari population (Asgari et al., 2013). Moreover,Yeniceus et al. demonstrated that the E3/E4 genotype is more prevalentin patients with RPL than in healthy controls (Yenicesu et al., 2010). Sim-ilar datawere obtained in a study byGoodman et al. who examinedApo Epolymorphisms in 69 RPL patients and 37 healthy controls and reportedthat Apo ε4 has a potential role in RPL (Goodman et al., 2009). A studybyOzornek et al. reported evidence of a relationship between Apo E poly-morphisms and RPL (Ozornek et al., 2010). By contrast, other studies re-ported that there is no association between Apo E polymorphisms andRPL (Bianca et al., 2010; Engin et al., 2013). In light of these studies anda recent meta-analysis (Meng et al., 2013), Apo E may be involved inthe pathophysiology of RPL. Furthermore, the substitution of argininewith cysteine at residue 158 (Apo ε2 allele) is reportedly associatedwith the increased affinity of Apo E2 for the low-density of lipoprotein(LDL) receptors (Stephens et al., 2005). This was also predicted by ourin silico analysis using PolyPhen-2 software (Fig. 1).

Apo E3 and Apo E4 bind the LDL receptors with equal affinity(Stephens et al., 2005). Therefore, the amino acid substitution at residue112 (ε4 allele) does not appear to affect the affinity of Apo E4 for the LDLreceptors, which is in accordance with our PolyPhen-2 analysis (Fig. 1).By contrast, mutation of the Apo ε4 allele reduces the affinities of ApoE4-containing lipoproteins for LDL receptors, and this reportedly under-lies the association of this allelewith the pathogenesis of RPL (Goodmanet al., 2009).

Considering these findings together, our data support the associa-tion of the Apo ε4 allele with RPL; however, in silico analysis predictedthat the amino acid substitution at residue 112 (Apo ε4 allele) is a be-nign mutation (Fig. 1). Accordingly, further studies are required to elu-cidate themechanism(s) underlying the link between RPL pathogenesisand the Apo ε4 allele.

Declaration of conflicting interests

There are no potential conflicts of interest for each author, concerningthe submitted manuscript.

Acknowledgments

The authors would like to express our sincerest appreciation to allthe subjects for participating in this study.

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