Analysis Of Single Nucleotide Polymorphism(SNP) Of Protein C Gene In A Patients With Thrombophilia And His Family

Background:Thrombophilia is the floorboard of many diseases of various thromboembolism due to genetic or acquired anticoagulation factors, coagulation factors, fibrinolytic defects or metabolic disorders. The word is put forward in1965for the first time by Egeberg who found a familial antithrombin III deficiency combined with embolism. Thrombophilic patients are often in hypercoagulable state that can induce thrombosis spontaneously.70%of thrombophilic patients have obvious clinical symptoms of thrombosis, while30%have no thrombosis.The clinical manifestations of thrombophilia are venous thromboembolism (deep venous thrombosis and pulmonary embolism), occlusive atherosclerosis, coronary heart disease and cerebral infarction and so on. Normally, the body of the anticoagulant system and coagulation system is in a dynamic equilibrium state. When the coagulation system was activated with an advantage, there will be multiple thromboembolism in the whole body (i.e. thrombophilia). There are mainly three systems play important roles in the anticoagulation:antithrombin Ⅲ (AT Ⅲ), protein C (PC) and tissue factor pathway inhibitor (TFPI). Protein C, protein S, thrombomodulin (TM) and activation of PC inhibitor (APCI) compose of protein C system play important roles in the process of anticoagulation. Protein C (PC) gene defects are the result of genetic risk factors for thrombophilia. There are extensive polymorphism in human genome, and the most simple form of polymorphism is single nucleotide substitution occurs in the genome, called single nucleotide polymorphisms. Through the study of PC in patients with SNP gene to make clear of the relationship between PC gene polymorphism and thrombophilia.Objective:This study aims to make clear of the relationship between PC gene polymorphism and thrombophilia through the discussion of a case of thrombophilic patients and their family members of the PC gene SNP.Methods:Collect the proband’s and his family members’venous blood sampling With0.109mol/L sodium citrate anticoagulation of1:9, and centrifugated at4℃for3500r/min for10minutes Immediately. Detection of PC:Ag and PC:A activity and other anticoagulant activity:Determination of PC:Ag use ELISA (Shanghai sun Biotech Corp), and PC:A was measured by chromogenic substrate method (Date), Sysmex1500Automatic Coagulation Analyzer. Extract the blood cells, and extract DNA with the DNA extraction kit from Promega company according to the instructions, and then stored at-80℃for use. According to the GenBank PC genomic DNA sequence reported, TransGen company synthesize the PCR primers, amplify the PC gene exons. Electrophorese the PCR products on1.5%agarose gel, the UV lamp cut specific PCR bands, and cut specific PCR bands in the UV lamp. Then recover and purify the corresponding PCR production using the DNA Purification kit with3S Spin agarose gel (Shanghai Shenergy gambling Biological Technology Co., Ltd.). Adopt the end labeled dideoxy method (AB13700, USA application of Bio System Inc) to procedure Gene sequencing.Results:1. Pc:Ag and Pc:A levels:among6members including the proband of the family, there are5manifestations of Pc:Ag and PC:A decreased, and drop to40%-50%the level of normal people; Pc:Ag and PC:A of the proband sister’s son are normal. Protein S:Ag (PS:Ag), protein S:A(PS:A), antithrombinⅢ: Ag (ATⅢ:Ag), antithrombinⅢ:A (ATⅢ:A) and anticardiolipin (ACA) of all the family members are normal. The function of liver and kidney probands and family members are normal. The proband smokes about20/day, but the other5members have no smoking habit.2. Analysis of protein C gene:when sequencing the PCR products of the proband protein C gene exon coding region and flanking sequences, we find that Exon second coding region nucleotide6711st existing base C deletion, insertion mutation in A base between6714th nucleotides and nucleotide6715th, so that theposition of Ala (alanine) and Asp (aspartic acid) was mutated to Glu (glutamic acid) and Asn (aspartic acid). Sequencing PC exon Ⅱthe other members of the family,4members were found have the same gene mutation with the proband.Conclusion:Compare and analyse the results of the Blast gene sequences through the website Pubmed, we find that there is a base C deletion in exon2of the coding region of the first6711nucleotides of protein C gene and a base A insertion mutation between the6714th nucleotide and6715th nucleotide. They are new mutational sites of hereditary protein C deficiency which decreasing the activity of protein C, and genetic factors in patients with thrombophilia.