The relationship between polymorphisms of P-selectin genes and plasma P-selectin concentration with thrombosis in non-valvular atrial fibrillation of Kazakh ethnicity

Maerjiaen Bakeyi, Zhi-Qiang Liu, Peng-Yi He, Lei Zhang, Yu-Chun Yang, Muhuyati

The First Affiliated Hospital of Xinjiang Medical University, Urumchi, Xinjiang 830011, China

Keywords:Soluble P-selectin Thrombosis Non-valvular atrial fibrillation

ABSTRACT Objective: To explore the relationship between polymorphisms in rs1800807 and rs1800808 locies of P-selectin gene and plasma concentrations of soluble P-selectin (sPs) and non-valvular atrial fibrillation (Af) coupled with thrombosis in Kazakh ethnicity. Methods: This was a case-control study, enrolling hospitalized Kazakh Af patients with and without thrombosis and healthy subjects. PCR-RFLP method was used to analyze the polymorphisms of the locies, and ELISA method was to measure plasma sPs concentrations. Subjects were divided into Af thrombosis, only Af and control groups. Results: We enrolled 42 Af thrombosis, 156 only Af and 307 control subjects. Carriers of GG genotype of the loci rs1800807 was the highest in Af thrombosis, followed by only Af and the lowest in the control groups (69 vs 23.7 vs 18.2%, P＜0.001). Carriers of TT genotype of the loci rs1800808 was the highest in Af thrombosis, followed by only Af and the lowest in the control groups (31.0 vs 13.5 vs 8.1%, P＜0.001). Plasma sPs concentrations was the highest in Af thrombosis group, followed by only Af group and the lowest in the control (52.20 vs 34.04 vs 35.85pg/mL, P＜0.001). Plasma sPs concentrations were significantly higher in the carriers of GG genotypes of the loci rs1800807 than in those of CC and CG genotypes (45.75 vs 33.86 vs 39.26pg/mL, P＜0.001) and also significantly higher in the carries of TT of the loci rs1800808 than in those of CC and CT (43.16 vs 36.74 vs 39.08pg/mL, P＜0.001). GG type of rs1800807 loci (OR=3.35, 95%CI: 2.03-5.55) and TT type of rs1800808 (OR=2.19, 95%CI: 1.65-2.90) showed significantly higher odd’s ratio for Af thrombosis, compared with the wild genotype and the highest tertile of plasma sPs aslo showed higher odd’s ratio (OR=1.31, 95CI%: 1.12-1.93), compared with the lowest tertile. Conclusion: polymorphisms of the locies of the PS gene were observed to exist in Af patients with thrombosis and with higher concentrations of plamsa sPs, suggesting their involvement in Af and thrombosis in Kazakh subjects.

1. Introduction

Atrial fibrillation (Af) is one of the common clinical disorder of electrical activity in the atria [1], with an age-standardized prevalence of 2.31% in general Chinese population [2], and as high as 34.5% in people who complain of arrhythmia symptoms from clinical setting [3].

An important and serious complication in long-term Af is the formation of thromboembolism, one of the main causes of Afassociated death and disability [4]. The risk of thromboembolism in patients with Af is 4-5.75 times higher than that of patients without Af [3,5]. The pathophysiological mechanisms of thrombosis in Af are diverse and complex and recent studies show that P selectin (Ps) and its regulatory genes play a certain role in this process [6,7]. The single nucleotide polymorphisms (SNPs) of the Ps gene generate changes in the concentration of sPs and increases the risk of thrombosis in different individuals and populations [8]. Furthermore, the circulating concentrations of soluble Ps (sPs) is associated with the polymorphism of the Ps gene and with the formation of thrombosis. It has been exhibited that the plasma sPs level of patients with nonvalvular Af coupled with cloudy shadow in the left chamber atrium or with a history of thrombosis in the left atrium are higher than that of non-Af subjects [9,10]

Based on current evidence, there are 13 genetic polymorphisms in the human gene encoding the Ps, whereas less attention has been attached to their association with Af and thrombosis. Previous studies have reported that the polymorphisms of rs1800807 and rs1800808 locies of the Ps gene are associated with Af and Afrelated thrombosis [11], which is also the same for the rs1800807 loci in Han and Uygur subjects [12]. Nonetheless, the association of the polymorphisms of locies with circulating sPs, Af and with Af-related thrombosis has not been confirmed in diverse population such as Kazakh ethnicity. Therefore, we aimed to explore the relationship of the polymorphisms of rs1800807 and rs1800808 locies of the Ps gene with plasma sPs, Af and with Af-related thrombosis Kazakh population.

2. Study subjects and methods

2.1 Study subjects

We consecutively enrolled Kazakh patients with Af coupled with and without thrombosis diagnosed with 24 hour Holter electrocardiography and echocardiography from the Cardiology Center of The First Affiliated Hospital of Xinjiang Medical University, using a case-control design based on the inclusion and exclusion criteria of the study. Healthy controls were enrolled from the health checkup center of the same hospital during the same period. The inclusion and exclusion criteria of the study are consistent with the previous study from our center [13]. In brief, inclusion criteria for Af patients: 1) confirmed consistent Af with medical history, conventional electrocardiography or 24 hour Holter electrocardiography; 2) confirmed to be non-valvular Af by transthoracic echocardiography. Inclusion criteria for Af coupled with thrombosis: confirmed thrombosis in the left atrium, or the silt-like changes in blood flow in the left atrium by transesophageal ultrasound or cerebral embolism caused by Af thromboembolism confirmed by senior neurologist with ＞10 year specialty experience on the basis of inclusion criteria for Af patients. Inclusion criteria for healthy controls: those confirmed to have no Af and thrombosis by electrocardiography and echocardiography. Exclusion criteria encompass autoimmune diseases, acute and chronic infections, malignant tumors, and acute coronary syndrome. This study was approved by the Ethic’s committee of the above-mentioned hospital. All the participants signed the consent before enrollment.

2.2 Study methods

Collection of basic information and blood samples: we collected data on age, gender, cigarette consumption and alcohol intake using before designed questionnaire and measured systolic and diastolic blood pressure, height and body weight using standard methods.

2.2.1 measurement of plasma sPs

We collected 5mL venous blood sample and measured plasma sPs concentration using ELISA methods with a reference range of 0-97pg/mL (The kit was purchased from Invitrogen company).

2.2.2 Genotype determination

Each genotype of SNPs for DNA samples were sequenced. PCR reaction conditions and primer sequence were given Table 1. Conditions for PCR reaction system is consistent with the previous study from the same team [13].

2.3 Statistical analysis

Gene counting method was applied to calculate and analyze each genotype and allele frequency. Hardy-Weinberg equilibrium analysis was used to test subjects’ representativeness. The number and percentage (n, %) were used to indicate the ratio of categorical data and chi-square test was used for among group comparisons. One way ANOVA test was used for the among group comparison of continuous variables such as plasma sPs concentration, followed by LSD test. Multi-variable logistic regression analysis was used to test the relation of SNP polymorphisms with Af related thrombosis and plasma sPs concentration, and presented as odd’s ratio (OR) and 95% confidence interval (95% CI). A P＜0.05 was defined as statistically significant. SPSS 22.0 package was applied for data analysis.

3. Results

3.1 Characteristics of study subjects

We finally enrolled 42 subjects with non-valvular Af and thrombosis, 156 subjects with non-valvular Af and 307 healthy controls and defined them as Af thrombosis, Af only and control groups. As in Table 2, There were no significant differences in the age, gender composition, cigarette consumption and alcohol intake among the three groups.

Table 1. Primer sequence

3.2 Results of Hardy-Weinberg equilibrium analysis

As in Table 3, the distribution of rs1800807 and rs1800808 sites satisfies the Hardy-Weinberg equilibrium test.

Table 3. Hardy-Weinberg test on the distribution of genotype frequency of rs1800807 and rs1800808

3.3 PCR products

As given in Figure 1-1and 1-2, in rs1800807, lattice 2 and 5 are heterozygous CG, with three bands. The 7th grid was GG complete mutant, with two bands. Grid 1, 3, 4, and 6 are wild CC genotype; in rs1800808, lattice 3 is the wild CC genotype with one band. The 4th grid is heterozygous CT, with three bands. Grid 1, 2, and 5 are mutant TT genotype.

Figure 1-1: (left) PCR product (size 189bp) and enzyme graphics of rs1800807 (right). Lattice 2 and 5 are heterozygous CG, with three bands. The 7th grid was GG complete mutant, with two bands. Grid 1, 3, 4, and 6 are wild CC genotype. The first grid of the left and right figures is the marker (size: 100-500bp,each interval:100bp).

Figure 1-2: (left) PCR product (size 154bp) and enzyme graphics of rs1800808 (right). Lattice 3 is the wild CC genotype with one band. The 4th grid is heterozygous CT, with three bands. Grid 1, 2, and 5 are mutant TT genotype. The first grid of the left and right figures is marker (size: 100-500bp, each interval: 100bp).

3.4 Comparison of Ps gene polymorphism and allele distribution among different groups

As in Table 4, the frequencies of the polymorphism of rs1800807 and rs1800808 loci of Ps gene were significantly different among groups (P＜0.001).

For rs1800807 loci, frequency of the GG homozygous mutation was the highestin the Af thrombosis group, followed by the Af only groupand by the control group (69 vs 23.7 vs 18.2%, P＜0.001). The GG homozygous genotype of the recessive model of rs1800807 gene locus was the highest in Af thrombosis, the second in the Af only group, and the lowest in the control group.

For rs1800808 loci, the frequency of the homozygous CC mutation the highest in the Af thrombus group, followed by the Af only group, and by the control group (31 vs 13.5 vs 8.1%, P＜0.001). Accordingly, the recessive model TT genotype was the highest in the Af thrombosis group, the second in the Af only group, and the lowest in the control group (P＜0.001).

3.5 Comparison of plasma sPs concentration among groups and genotype carriers

As in Table 5, the plasma sPs concentration was the highest inthe Af thrombosis group, the second in the Af only group, and the lowest in the control group (52.20±5.64 vs 49.04±5.82 vs 35.85±35.85±9.68 pg/mL, P＜0.001) and the difference was also statistically significant for between-group comparisons by LSD test (P for all ＜0.001). The plasma sPs concentration of GG genotype carriers of rs1800807 loci was significantly higher than that of CC and CG type carriers (46.38±9.57 vs 39.26±10.66 vs 35.75±8.18 pg/mL, P＜0.001). Similarly, the plasma sPs concentration of TT genotype carriers of the rs1800808 loci was significantly higher than that of CT and CC genotype carriers (52.26±11.21 vs 41.45±8.64 vs 36.74±7.75 pg/mL, P＜0.001).

Table 2. Characteristics of study population.

Table 4. Genotype and allele distributions (n,%)

Table 5. Comparison of plasma concentration of soluble P selectin in carriers of different genotype (pg/mL)

Table 6. Logistic regression analysis on the relation of rs1800807, rs1800808 and plasma P-selectin with atrial fibrillation combined with thrombosis

3.6 Relationship of Ps gene polymorphism with thrombosis and plasma sPs concentration in Af

Given in Table 6, logistic regression analysis showed that the GG genotype of rs1800807 was associated with increased OR for thrombosis (OR=3.35, 95%CI: 2.03, 5.55, P＜0.001); TT genotype of rs1800808 was associated with 2.19 fold increased odds for thrombosis (95%CI:1.65, 2.90, P＜0.001). The highest tertile of plasma sPs was associated with 1.31 fold increased odds for thrombosis (95CI%: 1.12,1.93,P=0.025), compared with the lowest tertile.

4. Discussion

Thromboembolism is one of the main causes of Af-associated death and disability [1,4]. Exploring the mechanism of thrombosis in Af and targeted treatment or intervention may be the effective ways to reduce Af-related adverse events and deaths. Main results of the current study encompass that the GG mutation of rs1800807 and the TT mutation genotype of rs1800808 have the highest carrier rate in patients with Af and thrombosis from Kazakh ethnicity; the plasma sPs concentration is the highest among carriers of the GG mutation genotype of rs1800807 and the TT mutation genotype of rs1800808 locies; rs1800807 The GG mutation genotype and the TT mutation genotype of rs1800808 locies are related to the increased risk of Af combined with thrombosis in Kazakhs.

Zeng et al and other scholars reported that the G allele frequency of the rs1800807 loci of the Ps gene is 0.463 and 0.344 in Af patients with and without thrombosis in Han population [14]. A previous study from our center also found that the polymorphism of the rs1800807 locus of Ps gene is associated with the thrombosis and serum sPs concentration in patients with Af from Han and Uygur population [12]. Current results from the rs1800807 loci are consistent with the above observations. In addition, the loci has been reported to be associated with the cardiovascular diseases, such as myocardial infarction and ischemic heart failure [15]. sPs exists in the granule membrane of platelet, which can be activated and adhered to the surface of activated platelets. The increase of plasma sPs concentration often causes thrombus formation; in turn, the circulating sPs will also increase during thrombosis, reflecting the activation of the hypercoagulable state of the circulatory system. Recent studies report that the polymorphisms of the sPs regulatory genes are significantly related to the increased circulating sPs concentration and the risk of venous thrombosis, whereas most studies have focused on deep vein thrombosis in the lungs or in the central nervous system, [16]. Therefore, current findings add evidence on the aspect that the mutations of sPs regulatory genes and changes in the circulating concentration of sPs may play a certain role in the formation of hypercoagulable state in condition of Af [17]. Another finding of this study is that the mutation of rs1800808 of the sPs gene is also associated with thrombosis and plasma Ps concentration in Af among Kazakh ethnicity, which is not consistent with previous studies reported by Zeng et al [18,19]. Nonetheless, current study design is unable to explain the observation, which needs to be further explored in studies with more suitable designs.

The merits of the current study encompass the case-control design and the strict inclusion and exclusion criteria and consistent results with previous studies. However, the study contains some limitations. First, the case-control design does not allow us to draw causal relations, whereas it is the most used methods for diseases with low prevalence such as Af. Second, some of the associations between diseases and exposure can be affected by environmental conditions, and or life styles [20], and therefore, whether the current observations can be applied to other populations remains to be studied. Third, Af is a disease with low prevalence, which makes the sample size lower in studies, and thus we still need larger sample size to validate the current observations,

In conclusion, the polymorphisms in the rs 1800807 and rs 1800808 locies of Ps gene may be associated the increased risk of thrombosis and elevated circulating sPs levels in Af patients from Kazakh ethnicity suggesting that these are potential vulnerable genes for thrombosis in Af.