Thrombophilia Pedigree Antithrombin Deficient Genetic Diagnosis

BackgroundHuman anticoagulant system has antithrombin system(Including antithrombinand heparin primarily inhibit the coagulation factor serine protease activity, such as:FXa, thrombin.) and protein C system (Including protein C, protein S,thrombomodulin, endothelial protein C receptor, the main inactivating coagulationfactor Ⅴ and Ⅷ etc.), The anti-thrombin antithrombin system is the body’s mostimportant anticoagulant factor, accounting for about70%of the anticoagulant activity.In1965, Egeberg O[1]. reported a familial antithrombin (AT) deficiency when firstproposed thrombophilia term, meaning increased tendency of thrombosis, followedby a long period time, it became synonymous with hereditary anticoagulant systemdysfunction. Griffin JH1981first reported by the genetic protein C (PC) deficiency;Then, in1984, discovered hereditary protein S (PS) deficiency[2-5]. At present theinternational community has reported AT mutations218kinds or more reported ATdeficiency pedigree than300cases; PC gene has been reported mutations262kindsor more, reports PC defect pedigree over300cases; reported PS mutations200kindsabove, PS defects reported over400cases of pedigree.Protein S (PS) is a liver, megakaryocytes and endothelial cells, the synthesis of vitamin K-dependent anticoagulant factors. A molecular weight of70kD, gene islocated on chromosome3with1Zone1(3p11.1-3p11.2), contains14introns and15exons, full-length gene sequence80kb. PS is a cofactor of activated protein canassist C (active protein C, APC) inactivating FVa and F Ⅷa factor, platelet surfaceAPC to assist phospholipid to form an immune complex of enzyme and substrate. PShas two coupling state and a free state in plasma. PS free state is able to play an activestate,35%of the total of PS; PS binding state can lose their activity and complementcofactor C4b-binding protein (C4b-Bp) combine to form a complex, a total of aboutPS65%. PS deficiency is a hereditary gene encoding a mutant PS occurrencecaused by an autosomal dominant genetic disease, which often causes repeated manyparts of venous thrombosis, such as mesenteric vein, femoral vein, peroneal vein, orpulmonary vein. The PS deficiency clinically divided into three types: I type is thelack of PS content, the molecular structure and molecular function properly; Ⅱtypeis unusual molecular structure PS qualitative change, can not play the anticoagulanteffect; Ⅲtype is reduced activity of free PS, the total content of normal PS.Protein C (PC) is a serine protease synthesized by the liver raw, vitaminK-dependent synthesis of raw materials needed to participate is a humananticoagulant system is an important anticoagulant factor. PC molecular weight of62kD, located in chromosome2, containing eight introns and exons9, full-lengthgene11.2kb. Protein S (PS) and protein C, thrombomodulin (or thrombomodulin) andthe endothelial protein C receptor like protein C system with the composition, thesystem functions involved in human anticoagulation plays an important anticoagulantsystem effect. Which is capable of activated protein C (active protein C, APC) stateof FVa and F Ⅷ a factor inactivation hydrolysis to reduce binding of FXa and FVaplay anticoagulant effect. PC deficiency is a hereditary gene mutations encoding thePC autosomal dominant genetic disease, can generate barriers and FVa APC and F Ⅷa fire barrier, causing venous thrombosis is common in deep venous thrombosis.Hereditary PC deficiency and hereditary PS deficiency can lead to abnormal PCsystems, often caused by multiple sites, repeated venous thrombosis, arterialthrombosis can also be seen, its clinical manifestations is very complicated. Antithrombin (AT) is a serine protease (serpins) inhibitors, mainly inhibit serineprotease activity of thrombin, FXa and other factors, the anticoagulant system in thebody plays an important role. Also called anti-thrombin antithrombin Ⅲ (AT Ⅲ), inthe early1950s, conducted scientific research discovered by Seegers, Johnson andFell, in turn named four AT (namely AT Ⅰ, AT Ⅱ, AT Ⅲ, AT Ⅳ).However, thereis currently only found in AT Ⅲ clinical significance, AT usually on behalf of thatAT Ⅲ[1]. Hereditary antithrombin deficiency is an inherited thrombophilia, and isdue to the gene encoding a mutated antithrombin caused by an autosomal dominantgenetic disease, the incidence in the population was extremely the two to five per tenthousandths[19-20], the Chinese people in venous thromboembolism incidence of5.75%-7.14%[18]. Antithrombin deficiency is one of the main factors leading tohereditary thrombophilia, most patients heterozygous for the gene mutation causes.Currently antithrombin gene mutation found in missense or nonsense mutationsaccount for56%splice site mutations,account for6%of base deletion, insertionmutations account for37%, gene rearrangements account for1%. Domestic researchantithrombin deficiencies thrombophilia is relatively small, so far reported11antithrombin deficiency pedigree. Hereditary antithrombin deficiency of the mainclinical manifestations were recurrent venous thrombosis, such as mesenteric vein,iliac vein, lower extremity deep vein, the most common sites of lower extremity deepvein thrombosis, can also lead to pulmonary embolism pulmonary thrombusformation in some cases; a handful of arterial thrombosis formation of myocardialinfarction, and cerebral infarction. Typically, AT defect population genetic factormutation in the AT gene, the risk of venous thromboembolism several times morethan the normal people, but mutations in the AT gene of the population will notcompletely the formation of hereditary AT deficiency, hereditary AT deficiencyoccurs is also affect incentives operation, trauma, such as long time, braking, elderly,malignant tumor, oral contraceptives, pregnancy, in the dual role of mutation andacquired cause hereditary gene, the incidence of hereditary AT deficiency willsignificantly increase.So far, thrombophilia pedigree domestic research reports are not many, thisstudy intends to two thrombophilia pedigree research, analysis of its phenotype diagnosis of antithrombin activity, activity of protein C and protein S activity,mutation analysis to detect the gene sequence, the patients to determine the type ofthrombophilia, and explore the gene diagnosis in the application in the diagnosis ofthrombophilia type.Objective1. The phenotype and gene diagnosis of2thrombophilia pedigree to disease andits members, and to explore the pathogenesis of family members.2. Antithrombin gene sequences of2member thrombophilia pedigree mutationanalysis, and comparison of human antithrombin gene mutation database, todetermine the mutation reported no antithrombin gene related sites.Methods1. The collection of two pedigrees, To collected blood samples of two familymembers.2. Using the chromogenic substrate method to detect the first families of9members and the second families of4members of AT activity (AT:A), protein Sactivity (PS:A), protein C activity (PC:A); to detect the AT antigen byimmunoturbidimetric assay (AT:Ag); simultaneous detection of the liver and kidneyfunction, blood coagulation, the two D-D dimer, lupus anticoagulant, anticardiolipinantibody.3. The molecular weight and content of AT with Western blot detection inplasma.4. Whole genome DNA was extracted from peripheral blood, after purification,using PCR for AT gene with7exons and their flanking sequences were amplified,amplified products to all members of the two families by direct sequencing and genemutation detection.5.100cases of normal screening to exclude polymorphisms mutations; Theprobands of two families of exon tenth of FV gene and exon fourteenth of prothrombin was amplified by PCR, sequenced, to rule out FV Leiden andprothrombin G20210A mutation.6. Image scanning, data processing, data analysis.Results1. The first family of the proband’s AT:A and AT:Ag were48%and121mg/L, theAT gene exon sixth was found10381T del, some members of the family of thedetected frameshift mutations in the same;2. The second families of the proband’s AT:A and AT:Ag were52.1%and266mg/L, projects and other members of the family of PC:A, PS:A, AT:A and ATcontent were normal, the sequence of AT gene by mutation analysis found noabnormal.ConclusionExistence of type Ⅰhereditary antithrombin deficiency of the first family todisease and some members, is a frameshift mutation caused by the AT gene of10381T del, the human antithrombin gene mutation database retrieval, the mutationhas been not reported; The second families do not have a mutation in the AT gene,diagnosis acquired thrombophilia with other detection.