SNAPC3 gene rs12093 polymorphism is significantly associated with ischemic stroke wind syndrome

Lian Gu ,Xian-Li Huang ,Yan Yan ,Yi-Bing Yang ,Lue-Jun Tang ,Li Su

1.The First Affiliated Hospital,Guangxi University of Chinese Medicine,Nanning 530023,China

2.Guangxi University of Chinese Medicine,Nanning 530001,China

3.School of Public Health,Guangxi Medical University,Nanning 530021,China

Keywords:Ischemic stroke SNAPC3 gene Gene polymorphism TCM syndrome Wind syndrome

ABSTRACT Objective:To investigate whether The small nuclear RNA activating complex polypeptide 3(SNAPC3) gene rs12093 polymorphism is associated with ischemic stroke (IS) and Traditional Chinese Medicine (TCM) syndromes of IS.Methods:This study enrolled 774 patients with ischemic stroke (IS) and 793 normal controls.Massarray technology was used for genotyping.And genetic association analysis was estimated by PLINK program.Results:①SNAPC3 gene rs12093 polymorphism was significantly associated with the risk of ischemic stroke wind syndrome of IS [OR=0.76,95%CI(0.58-0.99),P=0.047 in dominant model)].②After adjusting age and gender,SNAPC3 gene rs12093 polymorphism and the score of ischemic stroke wind syndrome(Padj=0.037 in additive model;Padj=0.009 in recessive model),the correlation is statistically significant.③After adjusting age and gender,platelet (PLT) (Padj=0.024 in addiction model;Padj=0.039 in dominant model)and thrombin time(TT)(Padj=0.042 in dominant model) were significantly associated with rs12093 polymorphism in patients with ischemic stroke wind syndrome.Conclusion:SNAPC3 gene rs12093 polymorphism may affect the occurrence of ischemic stroke wind syndrome.

1.Introduction

Ischemic stroke(IS) belongs to the category of "apoplexy" in traditional chinese medicine(TCM).Syndrome is a general summary of the etiology and pathogenesis of a certain stage of disease development.If the same disease has different pathological states at different stages of development,it will show different syndromes,that is,"same disease with different syndromes." The ancient physician Zhu Danxi put forward in his book "DanXiXinFa" that"there are insiders must be outside",the same disease has different syndromes,and there must be some material basis corresponding to the pathological manifestations.A number of previous studies have shown that gene polymorphisms are related to syndromes,and gene polymorphisms can be used as objective biomarkers of syndromes [1-3].Small nuclear RNA activation complex polypeptide 3 (SNAPC3) is located on chromosome 9p22.3,also known asSNAP50.SNAP50zinc finger domain plays an important role in promoter recognition and human small nuclear RNA gene transcription[4].There is no research report on the relationship betweenSNAPC3gene polymorphism and ischemic stroke.This study aims to explore the correlation between the polymorphism ofSNAPC3gene rs12093 locus and ischemic stroke and its TCM syndromes.

2.Materials and methods

2.1 Research object

2.1.1 Inclusion criteria

All subjects included in the case group met the diagnostic criteria of Western medicine and the diagnostic criteria of Chinese medicine.The diagnostic criteria of Western medicine need to be consistent with the diagnostic criteria of ischemic stroke in the"Key Points for Diagnosis of Various Cerebrovascular Diseases"revised by the Fourth National Cerebrovascular Disease Academic Conference of the Chinese Medical Association in 1995.All subjects were examined by cranial computed tomography (CT) and/or magnetic resonance imaging (MRI) medical imaging techniques for definitive diagnostic results.The diagnostic criteria of TCM also need to be consistent with the diagnostic criteria of stroke in the"Diagnostic Criteria for Stroke Diagnosis (Trial)" formulated by the State Administration of Traditional Chinese Medicine in 1996.Control group:The gender and age of all subjects included in the control group must match the case group.

2.1.2 Exclusion criteria

Case group:patients with ischemic stroke caused by traumatic brain injury,cerebral vascular malformation,arteritis,tumors,transient ischemic attack (TIA),infections,drugs,etc;petients with severe infection diseases or autoimmune system diseases;patients with severe heart,liver,kidney dysfunction and hematopoietic dysfunction.Control group:patients with heart,liver,kidney dysfunction and autoimmune system diseases.

All subjects in this study were self-reported as a Chinese Han population,and there was no genetic relationship between each other.This research plan had been submitted to the Medical Ethics Committee of Guangxi Medical University for review,and the research began after approval.Under the principle of informed consent,the research subjects had been informed in detail of the process and purpose of this research project.After all the subjects included in this study had fully understood the content of this study and expressed their consent,they signed the informed consent form.

2.2 Types of syndrome differentiation in TCM

According to the Stroke Syndrome Differentiation and Diagnosis Standards of Stroke Disease Diagnosis (Trial),the TCM syndromes of stroke in this study could be divided into 6 syndrome types,wind syndromes syndrome,fire syndrome,phlegm syndrome,syndrome of blood stasis,qi deficiency,yin deficiency and yang hyperactivity syndrome,respectively.Using this standard,patients with ischemic stroke were scored for TCM syndromes within 7 days of onset.Symptoms and signs with high specificity and sensitivity for the diagnosis of stroke syndromes were included in the scoring items.The score was based on its proportion to the diagnosis of syndromes.The total score was the score of the syndrome by adding the highest score of each item,and every syndrome ≧ 7 points means that the syndrome diagnosis is established.Each syndrome ≧ 7 points meant that the syndrome diagnosis was established.For each patient with ischemic stroke,the syndrome could be established after being scored by two TCM physicians strictly referring to the standard.If there was inconsistency between the two TCM physicians' syndrome scores,the third TCM physician with the title of associate chief physician or above would perform TCM syndrome scores and finally determined the syndromes.

2.3 General information

A total of 1567 subjects were enrolled in this study.The case group included 774 ischemic stroke patients (459 males,315 females),and the control group included 793 healthy controls (459 males,315 females).Case group subjects were from the Neurology Inpatient Department of the First Affiliated Hospital of Guangxi University of Chinese Medicine.During the same period,healthy controls were enrolled from the health check up center and hospitalized orthopedic patients with minor traumatismat the same hospital.The mean age of the cases and controls was 61.36±9.11years and 63.81±10.36years,respectively.No statistically significant differences were found between patients with IS and control individuals in gender (χ2=2.815,P=0.093) and age (t=-0.582,P=0.561).The population distribution of every TCM syndrome was shown as follows:the Wind syndrome (n=318),Fire heat syndrome (n=88),Phlegm syndrome (n=351),Blood stasis syndrome (n=177) and Qi deficiency syndrome (n=89),yin deficiency and yang hyperactivity syndrome(n=17).

2.4 Method

In the fasting state in the morning,2ml of peripheral venous blood was collected using EDTA anticoagulation tube.Genomic DNA was extracted by using the whole blood genomic DNA extraction kit from Beijing Aidlab biotechnologies CO.Ltd and strictly following its operating instructions.The optical density (OD) value was detected by NanoDrop2000 instrument and 1.25% agarose gel electrophoresis was uesed to detect the DNA quality.After the DNA quality was checked,it was transferred to -80℃ low-temperature refrigerator and stored for later use.Genotyping detection was performed by the MassARRAY SNP genotyping experiment technology in the Agena platform of Beijing Bomiao Biotechnology Co.,Ltd.,Beijing,China.

2.5 Detection of clinical parameters

Also,under the fasting state of all participants,2ml of anticoagulant blood was collected in the early morning.Using Mindray 6900 automatic blood cell analyzer to detect the level of platelets in whole blood.Coagulation function indexes of activated partial thromboplastin time (APTT),international normalized ratio (INR),D-dimer (DD),fibrinogen (FIB),thrombin time (TT),prothrombin time (PT) and Prothrombin time activity (PTA) were measured by the STAGO automatic anticoagulation coagulometer.

2.6 Statistical analysis methods

Genetic association analysis was carried out using PLINK software.The Chi-square test of goodness of fit was used to perform Hardy-Weinberg Equilibrium (HWE) test.Unconditional logistic regression analysis was used to assess the association between rs12093 polymorphism and the risk of ischemic stroke and its syndromes and the odds ratio (OR) and its 95% confidence interval (CI) was used to indicate the strength of the association.General linear regression was used to analyze the correlation between rs12093 polymorphism and quantitative traits in patients with ischemic stroke.The SPSS16.0 software was used to perform statistical analysis on other datas.The comparison of normal distribution measurement data between groups was analyzed by t test,and the comparison between count data groups was analyzed by chi-square test.All statistical test methods in this study were two-sided tests,and the test level was 0.05.

3.Result

3.1 HWE Test of SNAPC3 Gene rs12093 Polymorphism and Comparison of Genotype Frequency Distribution

The results of the chi-square test of goodness of fit showed that the genotype frequency distribution of theSNAPC3gene rs12093 in the control group was in line with Harvin equilibrium (PHWE=0.779).After the chi-square test,the results showed that the genotype frequency at the rs12093 locus was not statistically different between the stroke case group and the control group (P=0.268).As shown in Table 1,there was no statistically significant difference in the frequency distribution of rs12093 genotype between the IS wind syndrome,fire-heat syndrome,phlegm syndrome,blood stasis syndrome,and qi deficiency syndrome and the control group(allP>0.05).Since the number of samples of Yin deficiency and Yang hyperactivity syndrome in this study was too small (17 cases),the follow-up study and analysis of Yin deficiency and Yang hyperactivity syndrome types were not carried out.

3.2 Association Between SNAPC3 Gene rs12093 Polymorphism and The Risk of IS

As shown in Table 2,SNAPC3gene rs12093 polymorphism was not associated with the risk of IS in four genetic models (allP>0.05).After adjusting for age and gender,there was still no significant association (allP>0.05).

Table 2Associations of the rs12093 polymorphisms of SNAPC3 with Ischemic Stroke susceptibility

3.3 Association of SNAPC3 gene rs12093 polymorphism and TCM syndromes of IS

As shown in Table 3,multivariate logistic regression model analysis results showed that rs12093 polymorphism was significantly associated with the risk of IS wind syndrome [significant model:OR=0.76,95%CI (0.58-0.99),P=0.047)],After adjusting for gender and age,the association between rs12093 and the risk of IS wind syndrome [dominant model:ORadj=0.76,95% CIadj(0.58-0.98),Padj=0.040;additive model:ORadj=0.80,95% CIadj(0.64-0.99),Padj=0.047] was still statistically significant.In addition,before and after adjusting for age and gender,no no statistically significant difference was observed between rs12093 polymorphism and the risk of IS fire-heat syndrome,phlegm syndrome,blood stasis syndrome,and Qi deficiency syndrome (allP>0.05).

3.4 Association of SNAPC3 gene rs12093 polymorphism and the score of TCM syndromes of IS

As shown in Table 4,general linear model analysis results show that theSNAPC3gene rs12093 polymorphism was significantly associated with the IS wind syndrome score [additive model:β=-0.51,95%CI (-0.98--0.03),P=0.037);recessive model:β=-1.48,95%CI (-2.61--0.36),P=0.010)],after adjusting for gender and age,this correlation was still statistical significance [additive model:βadj=-0.51,95% CIadj(-0.98--0.03),Padj=0.037;recessive model:βadj=-1.49,95% CIadj (-2.61--0.37),Padj=0.009].In addition,before and after adjusting for age and gender,there was no significant correlation between rs12093 and the score of IS fire-heat syndrome,phlegm syndrome,blood stasis syndrome,and Qi deficiency syndrome (allP>0.05).

Table 3Associations of the rs12093 polymorphisms of SNAPC3 with different Ischemic Stroke TCM syndromes

3.5 Correlation between SNAPC3 gene rs12093 polymorphism and blood coagulation and platelet levels in patients with IS wind syndrome

As shown in Table 5,general linear model analysis results revealed that rs12093 was significantly associated with platelet levels in the patients with IS wind syndrome [additive model:β=-16.29,95%CI (-30.44--2.15),P=0.025);dominant model:β=-18.10,95%CI (-35.49--0.71),P=0.042)],thrombin time level [dominant model:β=-0.51,95%CI (-1.00--0.03),P=0.039) ].After adjusting for gender and age,the association between rs12093 and platelet levels in the patients with IS wind syndrome was still statistically significant[additive model:βadj=-16.30,95% CIadj(-30.36--2.24),Padj=0.024;dominant Model:βadj=-18.26,95% CIadj(-35.55--0.98),Padj=0.039],thrombin time level [dominant model:βadj=-0.50,95%CIadj(-0.99--0.02),Padj=0.042] .

Table 4Associations of the rs12093 polymorphisms of SNAPC3 with Ischemic Stroke TCM syndromes scores

Table 5Associations of the rs12093 polymorphisms of SNAPC3 with blood coagulation markers in patients with wind syndrome of Ischemic Stroke.

4.Discussion

The research scope of genomics is the entire genome level,mainly to study the location,structure,function of gene products and the relationship between genes.The use of genomics methods and technologies can screen out susceptibility genes related to the occurrence and development of diseases,which has important guiding significance for clinical prevention and treatment of diseases.Single nucleotide polymorphisms (SNPs) are one of the main research methods and technologies in genomics,and they are also an important method for studying complex diseases.In recent years,using SNPs detection technology have discovered multiple IS susceptibility gene loci.In which the chromosome 9p21.3 locus Studies have confirmed that there were susceptibility loci which associated with IS,cardiovascular disease,diabetes and other diseases[5].Several studies have reported that numerous loci(rs1333047-TT,rs1537378,rs10757278,rs2383206) on the 9p21 region of chromosome were associated with the risk of IS[6-8].It suggests that there may be a number of IS susceptibility loci in the 9p region of chromosome.SNAPC3,also known asSNAP50,is located on chromosome 9p22.3.There is no report on the association betweenSNAPC3gene polymorphism and IS.However,some scholars have found that the geneSYK(9q22.2) nearSNAPC3was related to the occurrence of atrial fibrillation in the genomewide association study of the Japanese population[9],and atrial fibrillation can induce the occurrence of stroke.What’s more,there were studies confirmed thatSYKrs2290890 T allele frequency andSYKgene rs2306040 (T/C) polymorphism are all associated with the risk of IS[10-11].These results suggest that theSNAPC3gene located at 9p22.3 may be related to the risk of IS.Therefore,this study explored the relationship between the geneSNAPC3and IS,and the results showed that the rs12093 polymorphism ofSNAPC3gene was not associated with the risk of ischemic stroke,but it was significantly associated with the risk of IS wind syndrome and its score.

The basic pathogenesis of TCM stroke can be summarized as"fire,phlegm,wind,deficiency,qi,and blood".The "wind" can be divided into "external”and "internal wind".For example,the theory of external wind advocated by sui and Tang dynasties,based on the theory of "Internal deficiency and pathogenic factors",thinks that apoplexy is caused by the invasion of external wind and the visceral dysfunction under the relatively weak state of qi and blood deficiency and unstable qi.According to the theory of "internal wind" after Tang dynasty,stroke is not caused by external wind,but caused by endogenous wind,which belongs to the disease of qi itself.[12] Li Shuiqin et al[13] have demonstrated that wind syndrome was the most common among the syndrome distribution of patients with stroke through the research of the syndrome of patients with acute IS.Therefore,"wind" is one of the main pathogenic factors of stroke.TCM syndrome is a summary of the physiological and pathological changes of the body at a certain stage of the development of a disease.Different seasons,regions,and personal physique have an important influence on the development of disease.When one disease is at different stages of development,ts physical and pathological changes are not the same,which is named "same disease with different syndromes."Differences between individuals are closely related to genetic polymorphisms.It means we can explore gene loci related to the formation of syndromes to reveal the essence of syndromes,which can provide molecular biological basis for TCM syndromes and "same disease with different syndromes." At present,some studies have found that there were inherent biomolecular basis of “same disease with different syndromes”.For example,Long Chunli et al[14] have found that the frequency ofMegsingene C2093T genotype was associated with IgA nephropathy spleen and kidney qi deficiency syndrome (P<0.05),but no statistical significance with Qi and Yin deficiency syndrome,liver and kidney yin deficiency syndrome and rheumatic heat toxin syndrome.Yan Xin et al[15] have found that IL-1B-31 TT type gene polymorphism and IL-10-1082 AG type gene polymorphism were related to the risk of spleen and stomach damp-heat syndrome of gastric precancerous disease,but there were no relation with liver and stomach discord syndrome,spleen and stomach asthenia syndrome;And IL-8-25 AA gene polymorphism was significantly related to the risk of precancerous gastric disease liver-stomach discordance syndrome and spleen-stomach weakness syndrome,but no significant association was found between IL-8-25 AA gene polymorphism and the risk of spleen-stomach damp-heat syndrome.What’s more,another study have found that TBX21 gene rs11657479 polymorphism was significantly associated with the risk of IS fire-heat syndrome,but no significant association was found between rs11657479 polymorphism and wind syndrome,phlegm syndrome,blood stasis syndrome,and qi deficiency syndrome[16].These findings indicates that it is feasible to find the intrinsic molecular biological basis of TCM syndromes through the detection of gene locus polymorphism,and it can provide theoretical support for TCM with the same disease and different syndromes.Our study found that theSNAPC3gene rs12093 polymorphism is significantly associated with the risk of IS wind syndrome and the score of wind syndrome,which indicates that theSNAPC3gene rs12093 polymorphism may be one of the basis of molecular genetics of the risk of IS wind syndrome and it probably can distinguish with other TCM syndromes.

Moreover,our study show thatSNAPC3gene rs12093 polymorphism was associated with PLT and TT in the patients with IS wind syndrome.Platelets and activation of coagulation system through various in vivo physiological and pathological responses can promote the formation of thrombus in vivo [17].Thrombosis leads to narrowing of the vascular lumen,and vascular blockage leads to stroke.Shen Mingqiang et al have found that Shen Mingqiang et al[18] found that GPIa C807T allele genotype was associated with the enhancement of platelet function in patients with acute stroke;Xu Xiuzhi et al[19] have found that βFg-455G/A polymorphism frequency had an effect on the platelet aggregation rate of patients with acute cerebral infarction.Another study have reported that the CALM1 gene rs3179089 polymorphism was significantly related to TT in patients with IS [20].These findings indicates that gene polymorphism has an effect on the coagulation function of patients with IS.It suggests that theSNAPC3gene rs12093 polymorphism may affect the coagulation function of patients with IS wind syndrome,but its underlying mechanism still needs further study.

SNAPC3gene rs12093 polymorphism may be related to the occurrence and development of IS wind syndrome,but no significant association are found between rs12093 polymorphism ofSNAPC3and other TCM syndromes.SNAPC3gene rs12093 polymorphism may be the molecular genetic basis of IS wind syndrome,which probably can provide support for TCM theory of "same disease with different syndromes".

Author’s contribution

Gu Lian:arrange experimental research,collect case samples and write articles;Huang Xianli:conduct experiments and statistical analysis of data;Yan Yan:clinical sample collection and data collection;Yang Yibing:data entry;Tang Luejun:conduct experiments;Su Li:The overall arrangement of experimental research work.