Introduction

Metabolic syndrome (MetS), as a cardiovascular risk factor, is considered a significant public health problem that is prevalent in developed and developing countries such as Iran [1, 2]. Etiopathogenesis of MetS is still not demonstrated, but previous studies have shown that a close relationship exists between MetS and oxidative stress, due to an imbalance between pro-oxidant and antioxidant species [3]. Selenoproteins with having selenium in their structure play a vital role in the protection against oxidative stress. Oxidative stress initiates by an excess of reactive oxygen and active nitrogen species [4]. In particular, previous studies have suggested that genotype variation in the selenoproteins’ genes are able to affect (a) selenium homeostasis and selenoproteins synthesis, (b) antioxidant defenses and redox control, and (c) endoplasmic reticulum (ER) signaling and degradation of misfolded proteins [5]. Recently, Selenoprotein S (SelS) and Selenoprotein P (SePP) have been considered as candidate proteins which may be related to cardiovascular disease and associated risk factors. SelS is involved in the processing and removing misfolded proteins such as cytokines and pro-inflammatory biomarkers. Also, it can affect the regulation of the cellular redox balance, and it may protect the endoplasmic reticulum against the deleterious effects of oxidative stress [6]. The human gene SEPS1 with seven exons, encoded as SelS with 189-amino acid protein, is located on chromosome 15q26.3 [7]. Previous research has indicated that genetic variation in the SEPS1 gene may be strongly associated with chronic inflammatory diseases [8,9,10]. Among various SEPS1 genetic variations, single nucleotide polymorphisms (SNPs) SEPS1 (rs28665122 C/T), promoter polymorphism and rs4965373 (3-untranslated region) have been revealed to be closely correlated with misfolded proteins in the ER, which may activate the transcription of several genes, especially those that encode pro-inflammatory cytokines [11]. Selenoprotein P (SePP) contains multiple selenocysteine residues per molecule (up to 10 in the human SePP) and not only plays a role in the selenium economy of the organism, but it also works as an antioxidant, protective protein for endothelial cells from oxidant molecules [12]. SEPP1 is located on chromosome 5q31 and consists of 6 exons [13]. Previous studies showed functional SNPs in SEPP1 rs3877899(UTR-3), and rs7579(UTR-3) are possibly related to several diseases [14, 15]. Our previous study revealed that the amount of SEPP and SelS decreased significantly among subjects who suffer from MetS [16]. So, we hypothesize that the presence of SNPs in their coding genes could be related to the decrease of SePP and SelS in subjects with MetS [17]. Therefore, we conducted a case–control study to investigate the associations between MetS and selected selenoprotein genetic polymorphisms in the SEPP1 and SEPS1, in an Iranian population. This study will help us to screen for MetS responsive genes and to better understand the molecular mechanism of MetS.

Materials and methods

Study population

The data used in this study was collected through the Selenegene study. This Selenegene Study is a local study which was performed to find the role of selenoproteins in MetS in subjects with a history of CAD. All subjects in this study were residents of the Isfahan Province, Iran, which is located in the central part of Iran. Patients were recruited sequentially during their angiography, myocardial revascularization or coronary artery bypass grafting (CABG) in the Chamran and Nour hospitals, which are tertiary university hospitals in Isfahan. An intervention was undertaken for recruitment which ran from December 2015 until the following March in 2016. Subjects with confirmed stenosis in one, two, or three vessels with angiographical documentation enrolled in the study. Details of inclusion and exclusion criteria are published in [16, 17]. The patients were interviewed to obtain their medical histories and then underwent laboratory assessments. Initial interviews and laboratory assessments included a questionnaire to collect demographic data, medical history, and detailed information for a nutritional profile including diet, selenium intake, and biochemical laboratory measurements. Information about age, sex, smoking habits, nutritional habits, history of CVD and related risk factors, along with the medication, were collected through interview questionnaires. The body mass index (in kg/m2) was calculated. Diabetes mellitus was defined as a plasma glucose ≥ 126 mg/dL, a self-report of a physician diagnosis of diabetes, or as the current medication use.

Sample collection

Fresh blood (5 mL) was collected from the antecubital vein of all subjects in the fasting state. The blood samples were used for isolation of DNA, and extracted DNA was frozen and stored at − 70 °C.

Genotyping analysis

DNA was isolated from peripheral blood lymphocytes using the standard salting out method [18]. Genotyping was carried out using TaqMan probes for allelic discrimination, as described by the supplier (LC480, Roche), and validated by capillary sequencing (AB3730, Applied Biosystems). One probe was complementary to the wild-type DNA strand and the other to the DNA strand with the mutation. Primers, annealing temperature and restriction endonucleases used in the study are listed in appendix 1. The reaction details are as follows: TaqMan PCR using an Eppendorf gradient type master cycler (Eppendorf, Germany) with a total volume of 12.5 mL, containing 6.25 mL. Taq PCR MasterMix, 0.5 mL each primer (10 mM), 1.5 mL genomic DNA and 3.75 mL H2O. The reaction conditions were: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s, and a final elongation step at 72 °C for 5 min.

SePP and SelS measurement

Serum SePP and SelS levels were justified using a commercially available human enzyme‐linked immunosorbent assay kit (Eastbiopharm, Hangzhou, China).

Biochemical Analysis

Total cholesterol, triglyceride, and HDL cholesterol were measured with the use of a Hitachi 902 Analyzer and using standard enzymatic kits (Parsazmun, Tehran, Iran). LDL-cholesterol concentrations were calculated using the Friedewald formula [19].

Definition of MetS

Subjects were selected based on the ATPIII criteria. That is when a subject has three of the five listed criteria, a diagnosis of MetS could be made. The primary clinical outcome of MetS was identified as cardiovascular disease. ATPIII defines MetS essentially as clustering of metabolic complications of obesity. The criteria listed including abdominal obesity, which is determined by increased waist circumference, raised triglycerides, reduced HDL-C, elevated blood pressure, and raised plasma glucose. Insulin resistance is not required for the diagnosis; however, most subjects meeting the ATP III criteria were insulin resistant [20].

Statistical analysis

A test of normality for the distribution of variables was performed using a Kolmogorov–Smirnov test. Data were expressed as mean ± SD. Differences between the groups were tested using the one-way ANOVA test or the Kruskal–Wallis test for continuous variables. The strength of association was presented as odds ratio (or 95% confidence interval) by using a logistic regression model. P < 0.05 was considered statistically significant.

Results

In this sub-study, 132 Iranian patients with cardiovascular disease were enrolled (71 MetS-affected individuals, 61 MetS-Unaffected individuals). Table 1 displays the demographic characteristics of CAD patients with MetS and without MetS. No significant differences were observed between either group with regard to age (55.6 ± 6.41 vs. 55.9 ± 7.52 P = 0.798), but a significant difference has been found with regards to gender prevalence (female, 32.3% vs. 15.5%, P = 0.021). Fasting blood sugar was higher among subjects with MetS (106.7 ± 14.1 vs, 96.3 ± 11.0, P < 0.001). Triglyceride level was higher among subjects with MetS (198.5 ± 122.0 vs. 139.1 ± 86.5, P = 0.003). Systolic and diastolic blood pressure, BMI, and waist circumference were higher among subjects with MetS (P = 0.05). There was no significant difference in the family history of CVD between the two groups (P = 0.460). Also, there were no significant differences with regards to smoking or nutritional habits (e.g., consuming beans, dairy, all types of meats, cereals, nuts, fruits, and vegetables) between the two groups (P = 0.9). The genotypic and allelic distribution of SEPP1 (rs3877899, rs7579) and SEPS1 (rs4965373, rs28665122) in subjects with and non-MetS is demonstrated in Table 2. Genotypes are in Hardy–Weinberg proportions (all P > 0.05, data not shown). Moreover, no significant differences were found in the genotype frequencies of SEPS1 (rs4965373, rs28665122), SEPP1 (rs7579, rs3877899) in patients with MetS and non-MetS group as the control group. A stepwise forward model was deployed, which shows that there was no significant relationship between presences of SNPs and MetS and the components of MetS (See Table 3). For finding an association, we used mutant isoform of rs4965373, rs3877899, rs7579 as the reference and for rs28665122, we used wild type as reference versus heterozygote type (Fig. 1).

Fig. 1
figure 1

Distribution of protein level of SePP and SelS based on the genotype in study participants

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Table 1 Clinical characteristics of Iranian population participants in the selenegene study
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Table 2 Frequency of SNPs in SEPS1 and SEPP1 in subjects with and without MetS
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Table 3 Association between SNPs in SEPS1 and SEPP1 with MetS and it’s components
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Table 4 demonstrates the expression of SEPS1 and SEPP1 genes in the protein level based on the genotype in the study participants. The mean of SelS in MetS subjects with SEPS1 (rs4965373) GG genotype was significantly lower than the non-MetS group (4496.99 ± 3688.5 vs. 6148.6 ± 1127.0, P = 0.009). Similarly, SEPS1 (rs28665122) TT genotype in the promotor region decreased expression of SelS significantly in the MetS group (2858.4 ± 500.700 vs. 6070.1 ± 1649.37, P = 0.019). The mean of SePP in MetS subjects with SEPP1 (rs3877899) GG genotype was significantly lower than the non-MetS group (40.73 ± 8.44 vs.83.91 ± 21.33, P = 0.002). The mean of SePP in MetS subjects with SEPP1 (rs7579) GG genotype was lower than the non-MetS group (55.52 ± 16.7 vs. 109.48 ± 29.78, P = 0.01).

Table 4 Expression of SEPS1 and SEPP1 based on the genotype in study participants
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Discussion

The results of this case–control study indicated no significant differences in the SEPS1 (rs4965373, rs28665122), SEPP1 (rs7579, rs3877899) allele frequencies between MetS and non-MetS subjects. However, the visible presence of the minor allele in rs4965373, rs7579, and rs3877899 could change the expression of SelS and SePP in the subjects with MetS and history of CAD. Our results, however, showed that the presence of polymorphism could not change the expression of SelS in the protein level. These are the first results that suggest a close relationship between these polymorphisms and susceptibility to MetS in subjects who suffer from CAD.

Mao et al. demonstrated that the selenium status is maintained better in pregnant women who carry the SEPP1 rs3877899 A allele [14]. Also, Previous studies confirmed polymorphisms in SEPP1 (SEPP1) rs3877899 and rs7579 can affect plasma selenium or selenoprotein concentrations or activity in response to supplementation [12, 18, 19].

The genotype SEPP1 rs3877899 is related to the decrement in the selenium concentration during pregnancy. These results suggest that presence the minor A allele can maintain selenium status better than can women with the G allele during pregnancy. These findings recommended that women carrying the rs3877899 minor A allele can better maintain their circulating selenium concentration during pregnancy and are more responsive to selenium supplementation, which related to the GPX3 activity [12]. These results could confirm our obtained results, which displayed presence of minor alleles of rs3877899 significantly decreased the level of SePP significantly in the MetS group (83.91 ± 21.33 vs. 40.37 ± 8.44, P = 0.002).

These exciting results confirmed that SePP, as a unique selenoprotein which contains various selenocysteine (Sec) residues per polypeptide (10 in human) is the main source and carrier of the selenium in plasma. It has been associated as an extracellular antioxidant, and in the transport of selenium to extra-hepatic tissues via apolipoprotein E receptor-2 (apoER2). So our obtained results is in line of previous results and confirmed that carrying the minor all of rs3877899 is able to decrease the antioxidant activity [14].

Regarding the influence of these genes on the inflammatory pathways in the progress of atherosclerosis and CAD being established [21], these proteins are essential for better understanding complex pathophysiology of CAD. In this regard, SEPS1 is a novel candidate gene which is involved in the regulation of inflammatory response [21]. Another distinct function of SEPS1 is the clearance of misfolded proteins from the endoplasmic reticulum into the cytosol to be broken up. This function is closely related to inflammatory and immune activities [10]. A common SNP in the promoter region of the SEPS1 gene, rs28665122 (−105G/A) could regulate the expression of SEPS1 and thereby affect the production of cytokines such as IL-1, TNFα, and IL-6, in addition to variance in the endoplasmic reticulum stress response [17]. Nonetheless, Martínez et al. explored six polymorphisms distributed through the SEPS1 gene (rs11327127, rs28665122, rs4965814, rs12917258, rs4965373, and rs2101171) in a large case control study and they could not demonstrate an association between SEPS1 polymorphisms and the increasing presence of inflammatory diseases [17].

Similarly, not only our results did not show any significant changes in the prevalence of different genotypes of rs28665122 (−105G/A) in both groups, but also there was no significant difference between the level of SelS protein based on this genotype between MetS and non-MetS.

Karunasinghe et al., exhibited serum selenium is related to SEPP1 rs3877890 GG and AG genotypes and oxidative stress in men (P = 0.0003–0.003) [23]. Interestingly, we have found SEPS1 (rs4965373) GG genotype increased expression of SelS significantly in the MetS group (1270.1 ± 6148.61 vs. 3688.45 ± 2331.09, 0.009).

SelS consider as a glucose-regulated endoplasmic reticulum-bound protein that plays a role in the inflammation and elimination of misfolded proteins from the endoplasmic reticulum. SelS is also regulated by inflammatory cytokines, and by ischemic circumstances. Presence of SNP in the promoter region decreases expression of SelS in humans, which are correlated with higher serum levels of inflammatory cytokines proposing a significant role for SelS in regulating inflammation. SelS by interaction by derlin-1 and p97 ATPase is able to eliminate misfolded proteins from the endoplasmic reticulum [24, 25].

This could explain the role of SelS in removing misfolded proteins, especially cytokines and how exciting and SNP in the promoter region of SEPS1 could decrease the expression of SelS in the MetS patients. Several reports previously described how the level of cytokines decreased in the MetS subjects and made them susceptible to obesity [26]. MetS is supposed to be related to a chronic inflammatory response, which is characterized by changing cytokine production and the activation of inflammatory signaling pathways [27]. In obesity, changes of adipokines and cytokines are supposed to provide to a low-grade inflammation within several secondary diseases such as MetS, insulin resistance, diabetes, arterial hypertension, and asthma [26,27,28]. However, another study which was conducted by Ogbera et al. suggested that there is no strong correlation between cytokines and MetS [29].

Recently, by performing a meta-analysis, Sun et al. revealed that the G-105A promoter polymorphism (rs28665122) in SEPS1 had been presented to increase pro-inflammatory cytokine expression. Therefore, suggesting that the protein product of this gene plays a role in inflammation [29] and, thus to be correlated with various types of human cancers and other diseases. Additionally, they found that according to the ethnicity-stratified sub-group analysis, SEPS1 rs28665122 polymorphism is significantly linked to increased risk of developing related diseases in Europeans but not among Asians [30]. Alanne et al., showed a significant association between minor allele of rs28665122 and ischemic heart disease [31].

Similar to our results, Hyrenbach et al. did not find any significant difference in SEPS1 allele frequencies between subjects who suffer from stroke and healthy controls, so they suggested that the SEPS1 -105A allele is not a significant risk factor for stroke [22]. Correspondingly, Park et al. used the case-cohort design and time-to-event analysis in FINRISK participants and showed that variation in the SEPS1 locus might affect CVD morbidity, especially in females [32].

Regarding SEPP1, we found that the existence of minor alleles in the (rs7579, rs3877899), which could significantly decrease the serum level of SePP, are functional and might be related to susceptibility to the pathogenesis of MetS, so changes in the expression of this gene may be due to genomic variation, which, perhaps, plays a role in the development of MetS. SePP, which is the most abundant plasma selenoproteins, is mainly responsible for the delivery of selenium to peripheral tissues and has antioxidant activities [23,24,25,26,27,28,29,30,31,32,33]. The most important activity of SePP is related to glucose metabolism in humans [34, 35] body mass, C-reactive protein, serum lipids, and carotid intima-media thickness, nonalcoholic fatty liver disease [35] in humans, Keshan beck disease, preeclampsia, prostate cancer, colorectal cancer, and aortic aneurism [36]. Genetic variation in SEPP1 has been reported to be associated with several metabolic phenotypes such as diabetes, but no study has assessed the relationship of these SNPs with MetS and CAD. Genomic variation in the SEPP1 (rs7579, rs3877899) were reported to have functional consequences on protein levels and\or function. Both of these variants also influenced the proportion of two SePP isoforms as well as response to the supplementation of selenium [23, 37]. The difference between genotypes disappeared after selenium supplementation. We conclude that functional polymorphisms in the SEPP1 gene influence the proportion of SePP isoforms (~ 60 and ~ 50 kDa) in plasma. Méplan et al. showed an elevation in the proportion of the 60-kDa isoform of SePP might increase selenoprotein synthesis and reduce the risk of disease [38]. Ishikura et al. illustrated that SePP inhibits vascular endothelial growth factor (VEGF)-stimulated cell proliferation, tubule formation, and migration in human umbilical vein endothelial cells [39].

Limitation

This study is limited to the small sample size, but as these variants evaluated in this patient groups for the first time, so it seems that considering the possible role of this proteins in these patients could be considered for more research. Also, we found that significant differences in sex frequency in both groups, which could affect the physiopathology of MetS and cardiovascular disease but due to the small sample size, it is impossible to compare the frequency of SNPs in the different sex groups with considering MetS.

Conclusion

In summary, the findings from the current study revealed no significant difference in the genotype frequency of the SEPS1 and SEPP1 variants in the MetS subjects with a history of cardiovascular disease. In the presence of genotype variation in the SEPS1 (rs28665122), SEPP1 (rs7579, rs3877899) significant changes has seen on the expression of the gene in the protein level between two groups and level of the SelS and SePP decreased in the MetS subjects.