A Forensic Pathological Research Of The Association Of SNPs With Thrombosis

Objective: Ascribing to its acute occurrence,high fatality rate,Pulmonary embolism is one of the most common fatal vascular diseases,which threat to human health and life seriously.It is acknowledgement that PE,usually secondary to deep venous thrombosis,is the direct cause of venous thromboembolism(VTE).Studies have shown that VTE has primary and/or secondary risk factors,of which about60% involved genetic risk factors.At present,there are many cases with secondary risk factors in nonfatal forensic pathological pulmonary embolism.However,there are still some difficulties in determining the coexistence of two types of risk factors' participation.It is helpful to detect and evaluate the genetic risk factors in the complex and difficult pulmonary embolism cases.At present,the forensic identification of primary risk factors for pulmonary embolism death cases is still lacking in China.In this study,we selected the fatal pulmonary embolism cases to study.Through analyzing the mutations of the relevant gene during thrombosis and their pathological changes.To research the primary risk factor screening system for complex pulmonary embolism,to provide molecular genetic evaluation indicators for this type of cases,and also provide a new theoretical basis for cause of death in forensic pathological pulmonary embolism identification.Methods:1 Sample selectionIn this study,we choose 16 pulmonary embolism cases as the experimental group.Selection criteria:1)VTE history,2)Autopsy and pulmonary embolism pathological diagnosis;12 non-pulmonaryembolism cases as the control group.Selection criteria:without VTE history and thrombosis in autopsy and pathologic diagnosis.2 Experiment method2.1 Pulmonary embolism related gene mutation detection.Whole genomic DNA is extracted from the sample venous blood with the kit in each group.Then the DNA was quantified with NanoQ,PCR amplificate of sample DNA,product purification and recycling.Four gene single nucleotide polymorphisms were selected: rs668(platelet endothelial cell adhesion molecular-1,PECAM-1);rs16984852(thrombomodulin gene,THBD);rs146922325,rs199469469(Protein C gene,PROC).Two exon:endothelial cell protein C receptor gene(EPCR)the fourth exon;Protein S gene(PROS)the tenth exon.The SNPs and exons were sequenced to detect the distribution and difference of the above-mentioned gene polymorphisms in the death cases of pulmonary embolism and control group.To analyze the variation rules,and initially explore the screening system for the gene locus of pulmonary embolism.2.2 Thrombosis pathological stainingAccording to rs668 and rs16984852 genotyping results pulmonary embolism group were regrouped.The thrombus tissues were fixed,dehydrated,transparent,embedded,and serially sectioned and processed for HE staining.CD34 and ?-SMA immunohistochemical staining were performed to observe the expression of CD34 and ?-SMA in different genotypes.3 Statistical analysisThe experimental data were statistically analyzed using SPSS 21.0statistical software,and the statistical significance of the two groups of experimental genotypes and allele distribution was analyzed by chi-square test;Logistic Regression Analysis of the relationship between SNP Genotypes and Pulmonary Embolism;One-factor analysis of variance(ANOVA)and the least significant difference were performed to compare mean of each group.P < 0.05 was supposed to be statisticallysignificant.Results:1 Pulmonary embolism related gene mutation detection.1.1 PECAM-1 rs668 genetype CC,CG,GG distribution frequency in PE group were: 2(12.5%),12(75%),2(12.5%);allelic genes C and G respectively were:50%.In the control group CC,CG,GG distribution frequency were: 2(16.7%),2(16.7%),8(66.6%);allelic genes C and G respectively were: 25.0% and 75.0%?There were statistically significant differences between the pulmonary embolism group and the control group(P<0.05).Logistic regression analysis of CG genotypes is associated with pulmonary embolism and can significantly increase the risk of pulmonary embolism(P < 0.05).1.2 THBD gene rs668 genetype GG,CT,TT distribution frequency in PE group were: 4(25%),12(75%),0;allelic genes G and T respectively were: 62.5% and 37.5%.In the control group GG,CT,TT distribution frequency were : 8(75%),4(25%)and 0;allelic genes G and T respectively were: 83.3% and 16.7%?The frequency of GT genotype was significantly higher than that in the control group.There were statistically significant differences between the pulmonary embolism group and the control group(P < 0.05).1.3 PROC gene rs146922325 genetype were CC in both PE group and control group;rs199469469 genetype were CTT insertion mutation.EPCR-4 and PS-10 exon sequencing showed no significant polymorphic variation sites.But we found CG genetype changes in the 132 base of EPCR-4 exon.There are 2 cases in PE group and 5 cases in control group,which were found this mutation.We also found 2 cases AG genetype changes in 54 base in control group.But there were no changes in the remaining groups.2 Thrombosis pathological staining2.1 HE stainingAll genotypes in the pulmonary embolism group were visible: mixedthrombus hyperplasia in the popliteal vein with unclear boundaries between the vascular wall and thrombi,thrombosis,new capillaries formation penetrating into the thrombus,and thrombosis partially recanalized.There were a large number of red blood cells and fibrin filling in the red thrombus in pulmonary arterial,which boundaries between thrombus and vessel wall was clear.2.2 Immunohistochemical staining of CD34CD34 positive expressed in both rs668 and rs16984852 genotype group of popliteal vein thrombosis.The CD34 positive cells in rs668 CG genotype group were significantly more than that in CC and GG genotype group,The CG genotype group was significantly different from the other two groups(P <0.05).The number of positive cells in rs16984852 GT genotype group was significantly more than that in GG genotype group.There was a significant difference between the two groups(P < 0.05).2.3 Immunohistochemical staining of ?-SMA?-SMA positive expressed in both rs668 and rs16984852 genotype group of popliteal vein thrombosis.The ?-SMA positive cells in rs668 CG genotype group were significantly more than that in CC and GG genotype group,The CG genotype group was significantly different from the other two groups(P <0.05).The number of positive cells in rs16984852 GT genotype group was significantly more than that in GG genotype group.There was a significant difference between the two groups(P < 0.05).Conclusions:1 PECAM-1 gene rs668 and THBD gene rs16984852 are the susceptible SNP in this experimental population.The CG and GT genotypes are the major risk genotypes and have play an important role in promoting PE.They may be as gene locus mutation screening site for genetic pulmonary embolism.The PROC gene rs146922325 and rs199469469 may not be the susceptibility SNP of pulmonary embolism in this population,EPCR-4 and PS-10 exons in this sample may not havepulmonary embolism susceptibility SNP.2 The rs668 CG and rs16984852 GT genotype may affected the organization and recanalization of thrombosis by increasing the number of fibroblasts and vascular endothelial cells during thrombosis.