The Ser290Asn and Thr715Pro Polymorphisms of the SELP Gene Are Associated with A Lower Risk of Developing Acute Coronary Syndrome and Low Soluble P-Selectin Levels in A Mexican Population †

Recent studies have shown that P-selectin promotes the early formation of atherosclerotic plaque. The aim of the present study was to evaluate whether the SELP gene single nucleotide polymorphisms (SNPs) are associated with presence of acute coronary syndrome (ACS) and with plasma P-selectin levels in a case-control association study. The sample size was estimated for a statistical power of 80%. We genotyped three SELP (SELP Ser290Asn, SELP Leu599Val, and SELP Thr715Pro) SNPs using 5’ exonuclease TaqMan assays in 625 patients with ACS and 700 healthy controls. The associations were evaluated with logistic regressions under the co-dominant, dominant, recessive, over-dominant and additive inheritance models. The genotype contribution to the plasma P-selectin levels was evaluated by a Student’s t-test. Under different models, the SELP Ser290Asn (OR = 0.59, pCCo-Dominant = 0.047; OR = 0.59, pCDominant = 0.014; OR = 0.58, pCOver-Dominant = 0.061, and OR = 0.62, pCAdditive = 0.015) and SELP Thr715Pro (OR = 0.61, pCDominant = 0.028; OR = 0.63, pCOver-Dominant = 0.044, and OR = 0.62, pCAdditive = 0.023) SNPs were associated with a lower risk of ACS. In addition, these SNPs were associated with low plasma P-selectin levels. In summary, this study established that the SELP Ser290Asn and SELP Thr715Pro SNPs are associated with a lower risk of developing ACS and with decreased P-selectin levels in plasma in a Mexican population.


Introduction
Acute coronary syndrome (ACS) comprises a spectrum of obstructive coronary artery diseases that most commonly arise from plaque rupture and/or erosion, leaving the vulnerable lipid-rich core exposed to the circulation. As a result, platelets and the coagulation cascade are activated, leading to acute thrombotic occlusion [1,2]. This syndrome is a consequence of atherosclerosis associated with a strong inflammatory component, which is immune mediated by chemokines. These molecules have an important role in the development of atherosclerotic plaque [3][4][5]. P-selectin is a chemokine, which mediates lymphocyte and monocyte recruitment, rolling, and diapedesis to the areas of inflammation [4][5][6]. Experimental studies have shown that higher expression of SELP increases adhesion, monocytes rolling to the vascular wall, accumulation of oxidized low-density lipoproteins, and the early formation of atherosclerotic plaque and other inflammatory diseases [4][5][6][7]. P-selectin contains 17 exons and is encoded by the SELP gene located on chromosome 1q21-q24 spanning <50 kb [8]. Recently, three single nucleotide polymorphism (SNPs) in the SELP gene in the exons 7, 12, and 13 [positions G1057A Ser290Asn (rs6131), G1980T Leu599Val (rs6133), and A2331C Thr715Pro (rs6136)] have been associated with myocardial infarction, hypertension, coronary heart disease, lupus erythematosus, type 2 diabetes mellitus (T2DM), and atherosclerosis [8][9][10][11][12][13]. Nonetheless, the association between these SNPs and other inflammatory diseases, such as diabetic retinopathy and multiple sclerosis is controversial, with negative results [14,15].
Considering the prominent role of P-selectin as a key in the chain of events leading to atherosclerotic plaque formation, the aim of this study was to investigate the association of three SELP SNPs (Ser290Asn, Leu599Val and Thr715Pro) with the risk of developing ACS. Furthermore, we evaluated whether these SNPs were associated with plasma P-selectin levels in a Mexican population sample.

Study Population
This case-control study was carried out at the Instituto Nacional de Cardiologia Ignacio Chavez. The sample size was calculated for unmatched cases and controls with OpenEpi software (http: //www.openepi.com/SampleSize/SSCC.html) with a statistical power of 80% and an alpha error of 0.05. Using this criterion, we included 625 patients with ACS (82% men and 18% women with a mean age of 57.97 ± 10.5 years) who were diagnosed based on clinical characteristics, electrocardiographic changes and biochemical markers of cardiac necrosis, according to guidelines from the European Society of Cardiology (ESC) and American College of Cardiology (ACC) [16,17]. The exclusion criteria were (1) patients with clear inflammatory pathologies on admission, such as infection established by clinical, laboratory, or image investigations, and (2) patients with an autoimmune disease or cancer previously diagnosed or documented during their hospitalization. Moreover, we included 700 healthy controls (66% men and 34% women with a mean age of 54.37 ± 7.65 years) coming from the Genetics of Atherosclerosis Disease (GEA) Mexican study previously described by Rosalinda-Posadas et al [18]. All healthy controls were asymptomatic and apparently healthy individuals without a family history of CAD and with a negative calcium score, indicative of the absence of subclinical atherosclerosis [18]. The exclusion criteria included not only the use anti-dyslipidemic, anti-hypertensive, and anti-diabetic drugs at the time of the study, but also congestive heart failure, as well as liver, renal, thyroid or oncological disease. All GEA participants were unrelated and of self-reported Mexican ancestry (3 generations). A Mexican mestizo was defined as a person who (1) was born in Mexico and (2) is a descendant of the original autochthonous inhabitants and of individuals (Caucasian and/or African, mainly Spaniards) who migrated to America in or after the XVI century. This study was conducted according to the principles of the Declaration of Helsinki and was approved by the Ethics and Research committee of our institution (registration number: 17CI09012010). Written informed consent was obtained from all individuals enrolled in the study.

Laboratory Analyses
After a 12-h overnight fast, EDTA blood samples were drawn and centrifuged within 15 min after collection; the plasma was separated into aliquots and immediately analyzed or frozen at −80 • C until analysis. Cholesterol and triglyceride plasma concentrations were determined by enzymatic/colorimetric assays (Randox Laboratories, UK). The phosphotungstic acid-Mg 2+ method was used to determine HDL-C concentrations. LDL-C was estimated in samples with a triglyceride level lower than 400 mg/dl, using the modified Friedewald formula [19]. Plasma lipid concentrations were determined within 24 h after blood sample collection. We followed the National Cholesterol Education Project (NCEP) Adult Treatment Panel (ATP III) guidelines and thus defined dyslipidemia with the following levels: cholesterol > 200 mg/dl, LDL-C > 130 mg/dl, HDL-C < 40 mg/dl, and triglyceride > 150 mg/dl (http://www.nhlbi.nih.gov/guidelines/cholesterol/atp3_rpt.htm). Type 2 diabetes mellitus (T2DM) was defined with a fasting glucose ≥ 126 mg/dL and was also considered when participants reported glucose-lowering treatment or a physician diagnosis of T2DM. Hypertension was defined by a systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg, or the use of oral antihypertensive therapy [18].

Genetic Analysis
DNA extraction was performed from peripheral blood in agreement with the method of Lahiri and Nurnberger [20]. The SELP G1057A Ser290Asn, SELP G1980T Leu599Val, and SELP A2331C Thr715Pro SNPs were genotyped using 5' exonuclease TaqMan assays on a 7900HT Fast Real-Time PCR system according to manufacturer's instructions (Applied Biosystems, foster City, CA, USA). In order to avoid genotyping errors, ten percent of the samples were determined twice; the results were concordant for all cases.

Determination of P-Selectin Levels
Samples were aliquoted and stored at −70 • C for further use. Plasma P-selectin levels were measured using a quantitative sandwich enzyme immunoassay technique (ELISA) kit in accordance with the manufacturer's instructions (Human P-Selectin/CD62P Quantikine ELISA Kit, R&D systems). The detection range was 0.8-50.00 ng/mL and the sensitivity was equal to the minimal detectable dose of this kit (≥ 0.121 ng/mL).

Statistical Analysis
All statistical analysis in this study was performed using SPSS version 18.0 (SPSS, Chicago, Il). Data of continuous variables were expressed as median and percentiles (25th-75th), while data of discrete variables [e.g., frequency (n, %)] were analyzed using Chi-squared or Fisher's exact tests. We used logistic regression tests to associate the SNPs with ACS under five inheritance models [16]. The correction of the p-values (pC) was performed with the Bonferroni test. Using the HAPLOVIEW version 4.1 software (Cambridge, MA, USA), we performed the haplotypes construction and linkage disequilibrium analysis (LD, D"). We tested whether our study population was in Hardy-Weinberg equilibrium (HWE) with a Chi-square test. Furthermore, we used the QUANTO software [http://biostats.usc.edu/software] to calculate the statistical power of our study and found it was 0.80. Using the Student's t-test, we analyzed the contribution of the genotypes on the P-selectin plasma levels. The values were expressed as means ± SD. The level of significance was set at p < 0.05.

Characteristics of the Study Population
Clinical and biochemical characteristics of the ACS patients and healthy controls are shown in Table 1. There were significant differences between the ACS patients and healthy controls. Compared to healthy controls, the ACS patients had a higher frequency of T2DM, hypertension, dyslipidemia, and smoking habit. Conversely, the total cholesterol, triglycerides, and LDL-C levels in ACS patients were lower than those in the control group; this effect may be due to their treatment with statins.

Allele and Genotype Frequencies
Genotype frequencies of the SNPs were in HWE. The frequencies of the SELP Leu599Val SNP was similar in ACS patients and healthy controls. Nonetheless, the SNPs [SELP Ser290Asn, and SELP Thr715Pro] were associated with a lower risk of ACS (Table 2). Under co-dominant, dominant, over-dominant, and additive models, the A (290Asn) allele of the SELP Ser290Asn SNP was associated with a lower risk of ACS (OR = 0.59, pC Co-Dom = 0.047; OR = 0.59, pC Dom = 0.014; OR = 0.58, pC Over-Dom = 0.061, and OR = 0.62, pC Add = 0.015, respectively). In the same way, under dominant, over-dominant, and additive models, the C (715Pro) allele of the SELP Thr715Pro SNP was associated with a lower risk of ACS (OR = 0.61, pC Dom = 0.028; OR = 0.63, pC Over-Dom = 0.044, and OR = 0.62, pC Add = 0.023, respectively). All models were adjusted for gender, age, blood pressure, BMI, glucose, total cholesterol, HDL-C, LDL-C, triglycerides, and smoking habit. Considering that the prevalence of T2DM (35%) and hypertension (57%) are highest in ACS patients versus healthy controls (10% and 29%, respectively), we performed a sub-analysis of the polymorphisms associated with a low risk of ACS (SELP Ser290Asn and SELP Thr715Pro). This analysis was made comparing individuals with and without T2DM and the other hand, individuals with and without hypertension. The results show that both polymorphisms were not associated with T2DM or with hypertension (Supplementary Tables S1 and S2). Therefore, this analysis corroborates that the genetic variation of these polymorphisms of the SELP gene are associated to the ACS, and not comes of the T2DM or hypertension.

Association of Polymorphisms with Plasma P-Selectin Levels
In order to define the functional effect of the SELP Ser290Asn and SELP Thr715Pro SNPs associated with a lower risk of ACS, we determined the plasma levels of P-selectin in individuals with different genotypes of these two polymorphisms. For this analysis, we included a subgroup of 30 healthy controls for SELP Ser290Asn (7 AA, 11 GA and 12 GG) and a subgroup of 30 healthy controls for the SELP Thr715Pro SNP (8 CC, 11 AC and 11 AA). In this study, we did not include the analysis of plasma P-selectin levels in patients with ACS, due to the fact that in the setting of the coronary syndrome, the comorbidities, such as insulin resistance/T2DM, hypertension, and inflammatory processes, as well as the use of the anti-dyslipidemic and/or anti-hypertensive drugs, may have altered the inflammatory markers levels, such as inflammatory cytokines, adhesion molecules, and C-reactive protein, masking the real impact of SELP polymorphisms on plasma P-selectin levels [23][24][25]. In this context, subjects carrying the AA (Ans/Ans) genotype of the SELP Ser290Asn SNP had a lower P-selectin plasma concentration (33.93 ± 9.79 ng/mL) than carriers of the GG (Ser/Ser) (44.76 ± 6.54 ng/mL, p =0.032) or GA (Ser/Ans) genotypes (48.04 ± 16.57 ng/mL, p = 0.049) ( Figure 1A). On the other hand, the analysis of the SELP Thr715Pro polymorphism showed that individuals with the CC (Pro/Pro) genotype had a lower concentration of P-selectin (26.44 ± 10.77 ng/mL) than AA (Thr/Thr) carriers (55.35 ± 14.05 ng/dl, p = 0.001). In addition, the individuals with the AC (Thr/Pro) genotype had lower P-selectin levels than AA (Thr/Thr) carriers (34.91 ± 14.46 ng/dl, p = 0.005) ( Figure 1B).

Functional Prediction
The functional prediction analysis showed that the presence of the A (Asn) allele of the SELP Ser290Asn polymorphism potentially produces a binding motif for Srp40 protein. In contrast, no evidence of potentially functional motifs was found for the SELP Thr715Pro polymorphism.

Discussion
In this study, we analyzed three relevant polymorphisms (Ser290Asn, Leu599Val, and Thr715Pro, respectively) of the SELP gene. The association of these SNPs with several inflammatory diseases in different populations is controversial, with positive and negative results [8][9][10][11][12][13][14][15]. In our study, the distribution of the SELP Leu599Val SNP was similar in both ACS patients and healthy controls. Nonetheless, the presence of the 290Asn and 715Pro alleles (SELP Ser290Asn and SELP Thr715Pro polymorphisms, respectively) was associated with a lower risk of developing ACS. In the same way, Reiner et al. reported in the CARDIA study that the SELP Ser290Asn and SELP Thr715Pro SNPs are associated with carotid intima-media thickness in young adults; however, these associations are different in European-American and African-American individuals [9]. In line with these data, Nasibullin et al. reported that the 290Ans allele of the SELP Ser290Asn SNP is associated with a lower risk of MI in a Russian population [13]. Similarly, the study of the risk of atherosclerosis in communities (ARIC), as well as the study of the Framingham heart (FHS) have shown that the genotype Pro715Pro is associated with a decreased risk of atherosclerosis in American and European populations [26,27]. In contrast with these data, in the ARIC study, Volcik et al. reported that the 290Ans and 715Pro alleles (SELP Ser290Asn and SELP Thr715Pro SNPs, respectively) were associated with the development of coronary heart disease in white but not in African Americans [11]. Similarly, Timasheva et al. reported that the 290Ans allele of the SELP Ser290Asn SNP is associated with the development of hypertension in ethnic Tatars originating from the Republic of Bashkortostan (Russian Federation) [12]. By the same token, Kou et al. reported that Thr715Pro or Pro715Pro genotypes of the SELP Thr715Pro polymorphism increased the risk of developing cardiovascular diseases (CVD) in a Chinese Han population [28]. Additionally, we found that the H3 (Pro-Leu-Ser) and H4 (Thr-Leu-Asn) haplotypes were associated with a lower risk of developing ACS, whereas H1 (Thr-Leu-Ser) was associated with a higher risk. As can be seen, the haplotypic combinations between SELP Thr715Pro and SELP Ser290Asn polymorphisms were not in linkage disequilibrium. Nonetheless, the protection haplotypes carry 715Pro and 290Ans alleles, and both of them were associated independently with a lower risk of cardiovascular diseases and other inflammatory diseases. This finding corroborated the role of these two alleles with the presence of ACS, whether they were analyzed independently or as haplotypes.
It is important to note that ACS patients and the healthy donors have much greater variation in blood glucose (102-188 versus 84-99) and diabetes mellitus (35% versus 10%). Considering these data, it is important to establish whether the polymorphisms are associated with T2DM or hypertension. In a sub-analysis, we showed that both polymorphisms were not associated with T2DM or with hypertension.
As can be seen, the associations of the SELP Ser290Asn and SELP Thr715Pro polymorphisms with ACS are contradictory in different study populations. We suggest that these discrepancies could be due to the classical cardiovascular risk factors and the environmental factors, such as diet, exercise, and lifestyle, which have an important role in the development of inflammatory diseases [29,30]. Another reason may be the fact that the allelic distribution of these polymorphisms varies according to the ethnic origin of the study populations. According to data obtained from the National Center for Biotechnology Information, populations from European, Asian, and African ancestry in Southwest US present a higher frequency of the A allele of the SELP G1057A Ser290Asn (rs6131) polymorphism (21.7%, 20.2% and 32.9%, respectively) when compared to Mexican mestizos and white American populations with a lower frequency of the A allele (9% and 14%, respectively). Concerning the SELP A2331C Thr715Pro (rs6136) SNP, Mexican mestizos, Europeans, and white Americans present a higher frequency of the C allele (8%, 8.8%, and 8.2%, respectively) than populations with Asian and African ancestry (0.2% and 2.5%, respectively) (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/), (https://www.ensembl.org/index.html).
We further determined the effect of the SELP gene polymorphisms on plasma P-selectin levels using genotype groups. We found that the AA (290 Asn/Asn) and CC (715 Pro/Pro) genotypes were associated with low P-selectin levels. As far as we know, this is the first study that showed the association of the SELP Ser290Asn and SELP Thr715Pro polymorphisms in P-selectin levels in individuals without the use of the anti-dyslipidemic or anti-hypertensive drugs. These drugs may modify the levels of the inflammatory markers, such as pro-inflammatory cytokines, adhesion molecules and C-reactive protein, masking the real impact of SELP gene polymorphisms on plasma P-selectin [23][24][25]. Nonetheless, the results concerning the association between P-selectin plasma levels and heart diseases are still contradictory. For example, Reiner et al. reported in the CARDIA study that the A (290Asn) and C (715Pro) alleles are associated with decreased plasma P-selectin levels and with the risk of developing atherosclerosis [9]. By the same token, Volcik et al. documented that the 715Pro allele is associated with lower P-selectin levels in the Atherosclerosis Risk in Communities (ARIC) study [27]. Similarly, Lee et al. determined that the lower serum levels of P-selectin decreased the risk of atherosclerosis [26]. At the same time, other reports have shown that the 715Pro (C) allele increased the expression of SELP mRNA, as well as the concentration of P-selectin levels in other inflammatory diseases, such as rheumatoid arthritis and T2DM [8,31]. As far as we know, the precise mechanism by which low and/or high P-selectin levels are associated with ACS remains to be elucidated. Nonetheless, recent data provide evidence that P-selectin upregulation on the endothelial cell surface mediates the effects of angiotensin II (Ang II), which has an important role in the development atherosclerosis [32]. In addition, Ang II stimulates not only the production of several molecules (adhesion molecules, chemokines, and cytokines) but also the oxidation and uptake of LDL, which promotes endothelial dysfunction [6,32]. On the other hand, Ang II triggers the synthesis of matrix metalloproteinases, the plasminogen activator inhibitor-1, and the proliferation of vascular smooth cells; this effect leads to the destabilization of atherosclerotic plaques [6]. Furthermore, using bioinformatics tools, we determined the potential effect of the SELP gene polymorphisms associated with ACS. The analysis of the SELP Thr715Pro polymorphism did not provide evidence of potential functional motifs. Nonetheless, the analysis of the SELP Ser290Asn polymorphism showed that the 290 Asn (A) allele generates a binding site for the Srp40 proteins. These proteins have multiple functions in the pre-mRNA splicing process, as well as in the regulation of alternative splicing, which leads to the production of protein isoforms [33,34]. In this context, we think that future investigations are warranted to understand the effect of these polymorphisms on P-selectin levels.
Some limitations should be considered. The P-selectin levels were only measured in a small sample of control individuals and experiments on RNA transcription or protein stability were not made. Considering these limitations, the effect of the SNPs on P-selectin plasma levels should be taken with care and studies in a large number of individuals are necessary to corroborate this association. In the same way, in our study it was not possible to determine the expression levels of P-selectin on the leukocyte's surface to confirm the data obtained in plasma.
In summary, this study demonstrated that the SELP Ser290Asn and SELP Thr715Pro polymorphisms are associated with a lower risk of developing ACS in a Mexican population. It was possible to distinguish two haplotypes (Pro-Leu-Ser and Thr-Leu-Asn) associated with a lower risk of developing ACS. On the other hand, both polymorphisms were associated with lower P-selectin levels in plasma. Lastly, due to the specific genetic characteristics of the Mexican population, we consider that additional studies will need to be undertaken in a larger number of individuals and in populations with different ethnic origins; these studies could help define the true role of these polymorphisms as markers of risk or protection from developing ACS and other cardiovascular events.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/2/270/s1, Table S1. Distribution of Ser290Asn and Thr715Pro SEL-P polymorphisms in individuals with and without T2DM. Table S2. Distribution of Ser290Asn and Thr715Pro SEL-P polymorphisms in individuals with and without hypertension.  Acknowledgments: This work was submitted in fulfilment of the requirements to obtaining the Doctoral degree of Gabriel Herrera-Maya in the PhD program of Biological Sciences of the Universidad Nacional Autónoma de Mexico (UNAM). Gabriel Herrera-Maya was supported by a fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACyT) Mexico City, Mexico with CVU number 545322. The authors are grateful to the study participants. Institutional Review Board approval was obtained for all sample collections. The authors would like to thank the technicians Silvestre Ramirez-Fuentes and Marva Arellano-Gonzalez for their participation in sample collection and DNA extraction.

Conflicts of Interest:
The authors declare no conflict of interest.

HDL-C
High-density lipoprotein-cholesterol LDL Low-density lipoprotein-cholesterol SELP P-selectin gene T2DM Type 2 diabetes mellitus SNP Single nucleotide polymorphism ACS Acute coronary syndrome