LI Liu-shui, WANG Fei, ZHOU Ao, YANG Qing, LIU Xian-jun?

1. Beijing Luhe Hospital, Capital Medical University, Beijing 101149, China

2. Shanghai Jinshan TCM-Integrated Hospital, Shanghai 201501, China

3. Beijing Chaoyang District Dongba Second Community Health Service Center, Beijing 100015, China

Keywords:

ABSTRACT Objective: To analyze the genotype and allele distribution characteristics of GPⅢa PLA2(rs5918), PEAR1 (rs12041331), and PTGS1 (rs10306114) genes related to the antiplatelet pharmacological effects of aspirin, providing reference for individualized treatment of Chinese Han NSTEMI patients.Methods: A total of 107 Han patients with NSTEMI in Beijing Luhe Hospital affiliated to Capital Medical University from January 2016 to December 2022 were selected as the research subjects.The genotypes of GPⅢa PLA2 (rs5918), PEAR1 (rs12041331)and PTGS1 (rs10306114) were detected by fluorescence staining in situ hybridization.The frequency distribution and allele distribution of genotype were analyzed.The results were analyzed whether there were statistical differences in the distribution of related alleles between the Han NSTEMI population and some populations in the 1 000 Genomes database.Results:In the Han NSTEMI population, the genotype frequencies of GPⅢa PLA2 (rs5918) locus were TT 97.20%, TC 2.80% and CC 0%, the allele frequencies were T 98.60% and C 1.40%.The genotype frequencies of PEAR1 (rs12041331) locus were GG 42.06%, GA 44.86% and AA 13.08%, the allele frequencies were G 64.49% and A 35.51%.The genotypes at the PTGS1(rs10306114) locus were all AA (100%), no AG or GG genotype was found.Conclusion: In the NSTEMI population of Han nationality, the mutation at GPⅢa PLA2 (rs5918) site related to aspirin antiplatelet pharmacology is rare, and there is no mutation at PTGS1 (rs10306114)site.Wild homozygotes are dominant in these two gene loci, while mutations in PEAR1(rs12041331) are more common.Some of the findings in this study are similar to those in previous reports or other populations included in the relevant database; however, some results differ from previous reports or other populations.

1.Introduction

Non-ST-segment elevation myocardial infarction (NSTEMI) is an acute coronary syndrome caused by the rupture and erosion of unstable atherosclerotic plaques in the coronary arteries.It is characterized by varying degrees of local thrombus formation and vascular spasm leading to vessel occlusion, resulting in acute myocardial ischemia and necrosis.The main clinical symptom is sudden chest pain that persists for a prolonged period.NSTEMI is more commonly observed in the elderly population and women compared to ST-segment elevation myocardial infarction (STEMI),and it is associated with a higher incidence of complications.[1]Diagnostic tests for NSTEMI show significantly elevated serum markers of myocardial necrosis and electrocardiographic changes indicative of acute myocardial ischemia, but without ST-segment elevation.NSTEMI is characterized by rapid disease progression,high mortality rate, and poor prognosis.In terms of pathogenesis,the rupture and erosion of unstable atherosclerotic plaques during NSTEMI activate the body’s self-protective mechanisms, leading to platelet adhesion, aggregation, and local thrombus formation,resulting in partial or complete occlusion of the coronary artery and subsequent acute myocardial ischemia and necrosis.In terms of treatment, both domestic and international guidelines[2-5] emphasize the importance of early, adequate, and sustained antiplatelet therapy in preventing disease progression and improving prognosis in NSTEMI patients.Aspirin is one of the representative drugs used in antiplatelet therapy for NSTEMI, but some patients exhibit aspirin resistance (AR), where they fail to respond to the normal pharmacological effects of aspirin and continue to experience cardiovascular events despite proper dosage and administration.

With the advancement of pharmacogenomics, it has been unequivocally established that the genetic polymorphisms of genes including GPⅢa PLA2, PEAR1, and PTGS1[6] are crucial determinants of individual variations in aspirin resistance or its antiplatelet efficacy.Consequently, tailoring therapeutic interventions based on individual genetic polymorphisms holds paramount clinical and economic significance.Currently, there is a paucity of systematic reports pertaining to the distribution patterns of the three genetic polymorphic loci of GPⅢa PLA2, PEAR1, and PTGS1 among the Han Chinese population with NSTEMI.This study focuses on the Han Chinese population afflicted with NSTEMI as the research cohort, undertaking detection analysis and investigation of three pivotal polymorphic sites within the GPⅢa PLA2, PEAR1, and PTGS1 genes, which are associated with the pharmacological effects of aspirin, in order to provide a theoretical foundation for the rational application of aspirin in NSTEMI patients with varying genotypes.

2.Materials and methods

2.1 Research object

This study employed a cross-sectional design and included a total of 107 Han Chinese patients with NSTEMI who received in patient treatment at Beijing Luhe Hospital affiliated to Capital Medical University, from January 2016 to December 2022.Among the participants, there were 80 males and 27 females, with an age range of 32 to 91 years and a mean age of 64.85±14.26 years.Informed consent for genetic polymorphism testing of the aforementioned loci was obtained from all participants.This study was approved by the Medical Ethics Committee of Beijing Luhe Hospital affiliated to Capital Medical University (Ethics Approval Number: 2023-LHKY-023-01).

2.1.1 Inclusion criteria

(1) Age 18 years with capacity for independent decision-making;(2) Both genders are eligible; (3) Han Chinese ethnicity; (4)Fulfillment of the diagnostic criteria for NSTEMI as stipulated in the “Diagnosis and Treatment Guidelines for Unstable Angina and Non-ST Elevation Myocardial Infarction”[7]; (5) Completion of genetic polymorphism testing for GPⅢa PLA2 (rs5918), PEAR1(rs12041331), and PTGS1 (rs10306114); (6) Availability of comprehensive clinical medical records.

2.1.2 Exclusion criteria

(1) Age ＜ 18 years old; (2) Non-Han ethnicity; (3) Failure to meet the diagnostic criteria for NSTEMI mentioned above, or suspected NSTEMI but not confirmed; (4) Incomplete testing of all three genetic polymorphic loci, GPⅢa PLA2 (rs5918), PEAR1(rs12041331), and PTGS1 (rs10306114), with only completion of 1-2 loci testing; (5) Incomplete clinical medical records.

2.2 Experimental reagents

The nucleic acid purification reagents, gene locus sequencing reaction universal reagent tubes, etc., were all purchased from Beijing Huaxia Shidai Gene Technology Development Co., Ltd.

2.3 Instruments

TL998A fluorescence detector (molecular diagnostic instrument),produced by Xi’an Tianlong Biotechnology Co., Ltd.; TD4A desktop low-speed centrifuge, produced by Changsha Yingtai Instrument Co.,Ltd.

2.4 Genetic polymorphic loci detection

The genetic polymorphic loci were determined using the fluorescence in situ hybridization method.The basic detection principle is as follows: based on the principle of base complementarity, known single-stranded nucleic acids (labeled with fluorophores) are used as probes to specifically bind to unknown single-stranded nucleic acids in the test sample, forming hybrid double-stranded nucleic acids (detectable).In the reaction system, 50 cycles of temperature amplification/cooling are performed.During temperature amplification, the double-stranded structure is denatured,and during cooling, the probe hybridizes with the unknown singlestranded nucleic acids in situ, successfully pairing and emitting fluorescence.Since DNA molecules are linearly arranged along the chromosomal axis, the probe can directly hybridize with the chromosome, thereby locating specific genes on the chromosome.The specific detection method is as follows: for each patient, 2-3 mL of venous blood samples were collected using disposable vacuum anticoagulant blood collection tubes (EDTA anticoagulant).The blood collection tube was gently shaken to mix the venous blood sample with the anticoagulant in the blood collection tube.Then,150 μL of blood sample was taken and added to 1 mL of red blood cell lysis solution, mixed and allowed to stand.After centrifugation at 3 000 rpm for 10 min, the supernatant was discarded.Then, 50 μL of nucleic acid purification reagent was added and mixed.Next,1.5 μL of the suspension was taken and added to the corresponding gene locus sequencing reaction universal reagent tube, mixed well.The mixture was placed in the appropriate position of the TL998A fluorescence detector (molecular diagnostic instrument).The microsequencing software program installed on the computer was opened, and the corresponding running parameters were set to detect the genotypes of the three genetic polymorphic loci, GPⅢa PLA2(rs5918), PEAR1 (rs12041331), and PTGS1 (rs10306114).

2.5 Hardy-Weinberg (H-W) equilibrium test

The theoretical frequencies of each genotype at the PEAR1(rs12041331) locus in the selected Han Chinese NSTEMI patient population were calculated, and a Hardy-Weinberg (H-W)equilibrium test[8] was performed to compare the theoretical frequencies with the observed frequencies of each genotype at this locus in the study.This test aims to determine whether the distribution of genotypes at this locus in the selected sample population reaches genetic equilibrium, thereby assessing the representativeness of the selected sample population for the Han Chinese NSTEMI patient population.

2.6 Genotype frequency and allele frequency calculation

The genotype frequencies of the GPⅢa PLA2 (rs5918), PEAR1(rs12041331), and PTGS1 (rs10306114) genetic polymorphic loci in the selected Han Chinese NSTEMI patient population were determined using a frequency counting method.For example, the genotype frequency of the heterozygous mutant genotype is calculated as the percentage of individuals with the heterozygous mutant genotype out of the total sample size, i.e.,heterozygous mutant genotype frequency = number of individuals with heterozygous mutant genotype / total sample size × 100%.The allele frequencies were then deduced based on the genotype frequencies.For instance, if only allele A and allele B exist at a genetic polymorphic locus, the frequency of allele A (P(A)) can be calculated as (number of individuals with AA homozygous genotype× 2 + number of individuals with AB heterozygous genotype × 1) /(total sample size × 2) × 100%.Similarly, the frequency of allele B(P(B)) can be calculated as P(B) = 100% - P(A).Subsequently, the distribution differences between the deduced allele frequencies and the corresponding allele frequencies in other populations recorded in the 1 000 Genomes database are compared.

2.7 Statistical analysis

The data in this research were statistically analyzed using SPSS 20.0 software.The count data were presented as frequencies(percentages).The distribution differences in genotype frequencies and allele frequencies at the same loci among different populations were compared using the chi-square (χ2) test.If more than 1/4 of the cells had theoretical frequencies less than 5, Fisher’s exact probability test was used.A significance level ofP＜0.05 was considered statistically significant.

3.Results

3.1 Hardy-Weinberg (H-W) equilibrium test results

Due to the observed frequency of 0 for the CC genotype individuals at the GPⅢa PLA2 (rs5918) locus and the observed frequencies of 0 for the AG and GG genotype individuals at the PTGS1(rs10306114) locus (as described later), the Hardy-Weinberg (H-W)equilibrium test was conducted based on the genotype frequencies at the PEAR1 (rs12041331) locus.The Hardy-Weinberg (H-W)equilibrium test was performed on the genotyping results of the PEAR1 (rs12041331) locus in 107 Han Chinese NSTEMI patients in this study.The results revealed no statistically significant difference between the observed frequencies and the expected frequencies for each genotype (GG, GA, and AA genotypes) at this locus (χ2=0.02,P=0.99 ＞ 0.05).This indicates that the selected sample population in this study is representative of the Han Chinese NSTEMI patient population, as shown in Table 1.

3.2 Genotype counts and genotype frequencies at each polymorphic locus

In this study, the genotype counts and genotype frequencies at the GPⅢa PLA2 (rs5918), PEAR1 (rs12041331), and PTGS1(rs10306114) genetic polymorphic loci in 107 Han Chinese NSTEMI patients are shown in Table 2.Among them, the most common genotype at the GPⅢa PLA2 (rs5918) locus is the wildtype homozygote (TT), accounting for approximately 97%, while the heterozygous mutant genotype (TC) is less common, accounting for less than 3%.No homozygous mutant genotype (CC) is observed at this locus.The heterozygous mutant genotype (GA) has the highest proportion at the PEAR1 (rs12041331) locus, approaching 45%,followed by the wild-type homozygote (GG), which accounts for around 42%.The homozygous mutant genotype (AA) has the lowest proportion, approximately 13%.At the PTGS1 (rs10306114) locus,all individuals have the wild-type homozygote (AA), accounting for 100%.No heterozygous mutant genotype (AG) or homozygous mutant genotype (GG) is observed.

3.3 Allele frequencies at each polymorphic locus

The allele frequencies at the GPⅢa PLA2 (rs5918), PEAR1(rs12041331), and PTGS1 (rs10306114) genetic polymorphic loci in107 Han Chinese NSTEMI patients in this study are shown in Table 3.At the GPⅢa PLA2 (rs5918) locus, the frequency of the T allele is approximately 99%, while the frequency of the C allele is only around 1%.At the PEAR1 (rs12041331) locus, the frequencies of the G and A alleles are approximately 64% and 36%, respectively.At the PTGS1 (rs10306114) locus, there is no G allele observed, and the frequency of the A allele is 100%.

Tab 2 Genotype count and frequency at each polymorphic locus (n, %)

Tab 3 Allele frequency at each polymorphic locus (%)

3.4 The corresponding allele frequencies in other populations included in the 1 000 Genomes database

Regarding the allele frequencies of the GPⅢa PLA2 (rs5918),PEAR1 (rs12041331), and PTGS1 (rs10306114) genetic polymorphisms in other ethnic populations, a search was conducted in the 1 000 Genomes database, followed by data compilation and summarization as presented in Table 4.This study included three East Asian populations, namely the Beijing Han Chinese (CHB),the Xishuangbanna Dai (CDX), and the Tokyo Japanese (JPT), as well as one European population, specifically the Iberian Spanish(IBS), one South Asian population, namely the Punjab Pakistani(PJL), one African population, represented by the African Southwest US (ASW), and one South American population, the Lima Peruano(PEL).

Among the investigated populations of different ethnic backgrounds,the T allele frequency at the GPⅢa PLA2 (rs5918) locus exceeded 85%, reaching 100% in the CDX population, over 99% in the CHB and JPT populations, and above 90% in both the ASW and PEL populations.In terms of the PEAR1 (rs12041331) locus, the G allele frequency exhibited considerable variability across different ethnic populations, ranging from 53% to 57% in the CDX, JPT, and ASW populations, while surpassing 85% in the IBS population.Across the diverse ethnic groups, the PTGS1 (rs10306114) locus demonstrated a generally high A allele frequency, approaching 100% in the PJL and PEL populations, and 100% in both the CHB, CDX, and JPT populations.

Tab 4 Some other populations’ related allele frequencies retrieved from 1 000 Genomes databases (%)

3.5 Comparison of the distribution difference of the corresponding alleles between the Han population of NSTEMI patients and the other populations

The distribution of allele frequencies for the GPⅢa PLA2(rs5918), PEAR1 (rs12041331), and PTGS1 (rs10306114) genetic polymorphisms among 107 Han Chinese NSTEMI patients in this study, as compared to the corresponding allele distributions in other populations included in the 1 000 Genomes database, is summarized in Table 5.In the Han Chinese NSTEMI patient population, there were no statistically significant differences in the T allele frequency(98.60%) at the GPⅢa PLA2 (rs5918) locus when compared to CHB (99.03%), CDX (100.00%), and JPT (99.04%) populations(all P ＞ 0.05).However, it was significantly higher than the PEL population (94.71%) (P ＜ 0.05), and significantly higher than the IBS population (86.45%), PJL (86.98%), and the ASW (91.80%)populations (all P ＜ 0.01).

In the Han Chinese NSTEMI patient population, there were no statistically significant differences in the G allele frequency (64.49%)at the PEAR1 (rs12041331) locus when compared to CHB (60.19%),JPT (56.25%), PJL (70.83%), ASW (56.56%), and PEL (70.00%)populations (all P ＞ 0.05).However, it was significantly lower than the CDX population (53.23%) (P ＜ 0.05), and significantly lower than the IBS population (85.51%) (P＜ 0.01).

In the Han Chinese NSTEMI patient population, the A and G allele frequencies at the PTGS1 (rs10306114) locus were 100.00%and 0%, respectively, which were the same as CHB, CDX, and JPT populations.However, the A allele frequency was significantly higher than the IBS (93.93%) and ASW (86.89%) populations (bothP＜ 0.01), and there were no statistically significant differences when compared to the PJL (98.96%) and PEL (97.65%) populations (all P＞ 0.05).

Tab 5 Comparison of the distribution differences of related alleles between the Han population of NSTEMI patients and some other populations (χ2 value)

4.Discussion

In this study, a sample population of 107 Han Chinese NSTEMI patients was selected.The Hardy-Weinberg (H-W) equilibrium test confirmed that the population was representative and exhibited good representativeness.The genotype and allele distribution characteristics of three important polymorphic loci (GPⅢa PLA2,PEAR1, and PTGS1) related to the pharmacological effects of aspirin on platelets were systematically analyzed in this sample population.

The activation of platelet membrane glycoprotein (GP) IIb/IIIa receptors is the ultimate common pathway leading to platelet aggregation, coronary artery thrombosis formation, and myocardial ischemia.[9] The GP IIb/IIIa receptor, also known as integrin αIIbβ3, is encoded by the highly polymorphic GP IIIa subunit gene.The most common allelic variants are PLA1 (T) and PLA2(C).It has been reported that carriers of the PLA2 (C) allele have a significantly increased risk of myocardial infarction or unstable angina.[10] In this study of 107 Han Chinese NSTEMI patients, the wild-type homozygous genotype (TT) was predominant at the GPⅢa PLA2 (rs5918) locus, accounting for 97.20%, while the mutant heterozygous genotype (TC) accounted for only 2.80%.The mutant homozygous genotype (CC) was absent.This result is similar to the findings of Xu JD et al.[11] where, in their study of 83 hospitalized patients receiving enteric-coated aspirin treatment, the PLA1/PLA1 (TT) genotype was found in 80 cases (96.39%), the PLA1/PLA2 (TC) genotype in 3 cases (3.61%), and the PLA2/PLA2(CC) genotype was absent.There were no statistically significant differences in the distribution of genotypes compared to our study(χ2=0.01,P=0.92 ＞ 0.05).Accordingly, in the 107 Han Chinese NSTEMI patients, the predominant allele at the GPⅢa PLA2(rs5918) locus was T, accounting for 98.60%, while the allele C accounted for only 1.40%.The allele distribution at this locus was similar to that of three East Asian populations including CHB, CDX,and JPT.However, the T allele frequency was higher than that of the PEL population in South America and significantly higher than that of the IBS population, the PJL population in South Asia, and the ASW population, which may be related to factors such as race or geography.

Platelet Endothelial Aggregation Receptor 1 (PEAR1) belongs to the receptor tyrosine kinase family and is involved in contactinduced activation resulting from platelet interactions.[12-13] The PEAR1 gene is primarily located on human chromosome 1q23.1,and its single nucleotide polymorphisms are closely associated with the occurrence of certain diseases and individual variations in antiplatelet pharmacological effects.[14] For example, the PEAR1(rs12041331) GG genotype shows a good response to aspirin, the GA genotype has a moderate response, while the AA genotype exhibits resistance to aspirin, thus having the poorest response and affecting patient prognosis.[15] In this study, the Han Chinese NSTEMI patient population showed a higher prevalence of mutations at the PEAR1(rs12041331) locus, with the wild-type homozygous genotype (GG),mutant heterozygous genotype (GA), and mutant homozygous genotype (AA) accounting for approximately 42%, 45%, and 13%respectively.In a study by Wang LY et al.[16] involving 183 patients with acute myocardial infarction (AMI), the distribution of the three genotypes was 55 cases (30.05%), 91 cases (49.73%), and 37 cases (20.22%).Compared to our study, there was no statistically significant difference in the distribution of this genotype between the two groups (χ2=5.11, P=0.08 ＞ 0.05).However, the genetic polymorphic loci detected in their study were not at the rs12041331 locus of the PEAR1 gene but at the rs2768759 locus on the same gene.This suggests that the genetic polymorphisms of multiple loci on the PEAR1 gene may be associated with susceptibility to myocardial infarction.In the Han Chinese NSTEMI patient population of this study, the allele frequencies of G and A at the PEAR1 (rs12041331) locus were approximately 64% and 36%respectively.These frequencies showed no statistically significant difference compared to the corresponding allele frequencies (62.32%and 37.68%) reported by Shi XJ et al.[17] in patients with ischemic diseases (χ2=0.24, P=0.62 ＞ 0.05).Furthermore, the distribution of allele frequencies at this locus in the Han Chinese NSTEMI patient population showed no difference compared to that of CHB, JPT,ASW, and PEL populations.Therefore, it appears that there is no correlation between the genotype or allele distribution at the PEAR1(rs12041331) locus and certain diseases (such as NSTEMI and ischemic diseases) or racial differences mentioned above.

Prostaglandin-endoperoxide synthase 1 (PTGS1), also known as cyclooxygenase-1 (COX-1), is the main target of aspirin’s antiplatelet pharmacological effects.Since COX-1 is encoded by the PTGS1 gene, genetic polymorphisms in the PTGS1 gene can affect the activity of COX-1, thereby influencing platelet aggregation and the antiplatelet pharmacological effects of aspirin.Therefore, there is a certain correlation between PTGS1 gene polymorphisms and the occurrence of aspirin resistance.[18] It has been reported that patients with the PTGS1 GG genotype have a 16.495-fold higher probability of developing aspirin resistance compared to patients with the AG/AA genotypes (95% CI: 4.076 - 66.754), making the PTGS1 GG genotype one of the risk factors for aspirin resistance.[19]In the population of different ethnicities investigated in this study,the frequency of the A allele at the PTGS1 (rs10306114) locus was generally high, exceeding 86% in all populations, and was 100% in the CHB, CDX, and JPT populations, as well as close to 100% in the PJL and PEL populations.In the Han Chinese NSTEMI patient population, the PTGS1 (rs10306114) locus was exclusively AA genotype, with no presence of AG or GG genotypes.The frequency of the A allele in this population was also 100%, similar to the findings in a study by Xiao F et al.[20] involving 71 Han Chinese patients using aspirin.In a study by Chen L et al.[21] involving 1175 patients using aspirin, only 1 case (0.09%) had the AG genotype at the PTGS1 (rs10306114) locus, while the remaining 1174 cases(99.91%) were AA genotype.The detection of the AG genotype in their study may be due to a larger sample size or the inclusion of non-Han Chinese patients (however the ethnicity of the patients was not described).

In summary, this study investigated the genotype and allele distribution characteristics of three important loci, GPⅢa PLA2(rs5918), PEAR1 (rs12041331), and PTGS1 (rs10306114), related to aspirin’s antiplatelet effects in the Han Chinese NSTEMI patient population.It was found that the wild-type homozygous genotype was predominant at both the GPⅢa PLA2 (rs5918) and PTGS1 (rs10306114) loci, with rare mutations observed.However,mutations were more common at the PEAR1 (rs12041331) locus,with a higher proportion of mutant heterozygous genotypes.For patients with NSTEMI or those at high risk of developing NSTEMI,it is recommended that genetic testing of relevant loci be conducted when feasible.This would facilitate individualized antiplatelet therapy based on genotype results when necessary.

Author’s contribution

Li Liushui: Responsible for experimental design, sample testing,data processing, and manuscript writing.

Liu Xianjun: Responsible for manuscript revision and experimental guidance.

Wang Fei, Zhou Ao, and Yang Qing: Responsible for manuscript revision and proofreading.

All authors declare no conflict of interest.