Study On The Pathogenesis Of Congenital Dysfibrinogenemia Caused Bymutation Of FGG Genes

Part one:Study on molecular pathogenesis of congenital dysfibrinogenemia caused by ? Ala327 Val heterozygous mutationBackground & objective:Fibrinogen(Fg)is a glycoprotein with molecular weight of 340 KD,which participates in the formation of fibrin clot and platelet aggregation,and plays an important role in maintaining the balance of hemostasis.Mutations in the gene encoding fibrinogen can cause abnormalities in fibrinogen structure and function,leading to congenital dysfibrinogenemia(CD).The clinical manifestations of the disease is complex,most patients do not have any clinical manifestations,some patients with thrombosis or bleeding,and some patients with bleeding and thrombosis coexist.Female patients with menorrhagia,recurrent abortion and other symptoms.At present,a novel heterozygous mutation(FGG c.1058 C > T)has been found in an asymptomatic patient with abnormal fibrinogenemia.The FGG c.1058 C > T mutation is located in exon 8 of the FGG gene and mutates into ?Ala327Val.We extracted and purified fibrinogen from the plasma of the patient and four other members of her family and performed a series of studies on fibrinogen function,including fibrinogen aggregation test,fibrinogen clot lysis test,thromboelastogram test,and observed the network structure in the proband's fibrin clot by scanning electron microscope,to study the possible effects of mutation on fibrinogen structure and function.The pathogenesis of congenital dysfibrinogenemia caused by FGG c.1058 C > T,? Ala327 Val heterozygous mutation was studied.Methods:(1)The basic data of patient and her family members were collected and routine blood coagulation tests including APTT,PT,TT,Fg(Clauss method and Immune turbidimetry),liver and kidney function tests and routine physical examination were performed;(2)extracting DNA from peripheral blood cells of CD patients,designing and synthesizing primers in the upstream and downstream of exons,using PCR technology to amplify all exons of fibrinogen gene,detecting the base sequence of the amplified fragment.The sequencing results were compared with the standard sequences in the NCBI gene database to find the gene mutation types of CD patients;(3)the plasma fibrinogen aggregation of CD patients and normal controls was monitored dynamically to understand the function of fibrinogen aggregation in CD patients;(4)The fibrinolytic process of patients with CD and normal controls was dynamically monitored to determine whether fibrinolytic prolongation or fibrinolytic resistance existed in the fibrinolytic process of patients with CD;(5)monitoring the coagulation function of proband and her family members with thrombomodometer;(6)plasma fibrinogen was extracted and purified from proband and her family members,and SDS-PAGE electrophoresis was performed to determine whether fibrinogen had variant bands;(7)observe the network structure in the proband's fibrin clot by scanning electron microscope;(8)Analysis of the effect of mutation sites on fibrinogen by amino acid modeling.Through the above methods,the molecular mechanism of congenital dysfibrinogenemia caused by ? Ala327 Val mutation was discussed.Results:(1)There was no history of abnormal bleeding or thrombosis in the proband and her family members.The plasma fibrinogen activity concentration(Clauss method)was significantly lower than the fibrinogen antigen concentration(Immune turbidimetry)in the proband and her family members.The results of liver and kidney function tests were normal.(2)the exon 8 of FGG gene in the proband and her family members were found to have FGG c.1058 C > T heterozygous missense mutation by gene sequencing,resulting in ?Ala327Val heterozygous mutation;(3)the results of fibrinogen aggregation test showed that the change of OD value of fibrinogen aggregation curve in proband and her family members was smaller than that in normal controls,and the maximum OD value(average 0.283Abs)was lower than that in normal controls(0.422Abs),suggesting that the aggregation rate of fibrinogen in proband and her family members was slower than that in normal controls.There's a defect in fibrin aggregation.(4)the results of fibrinolysis curve showed that the maximum OD value of the proband and her family members was lower than that of the normal control,and the turbidity of the clot began to decrease after 496 seconds on average(control: 660 seconds).The results showed that the proband and her family members had no delayed fibrinolysis and resistance to fibrinolysis compared with the normal control;(5)the results of thromboelastography(TEG)showed that the K value of proband and her family members increased,the average value was 3.7min(normal control was 2.3min),the corresponding Angle value decreased,the average value was 52.8 °(normal control was 61.5 °),indicating that the fibrinogen function of proband and her family members was low.The rate of clot coalescence is reduced;(6)The results of SDS-PAGE electrophoresis showed that there was no change in the mobility of fibrinogen in the proband and her family members.(7)The structure of fibrin clot network of proband was observed by scanning electron microscope(SEM),which showed that the size of fibrils were different,the arrangement of fibrils was irregular,and the ends of fibrils curled into clusters.The spatial structure of the fiber network is loose,the network aperture increases,and the number of fiber branching nodes is more than that of the normal control;(8)Amino acid modeling analysis showed that ?Ala327Val mutation caused fibrinogen D region changes,which affected the structural stability of fibrinogen region.Conclusion:1.Based on the data of the proband and the members of the family,we diagnosed that the family had congenital dysfibrinogenemia,and Ala327 Val heterozygous mutation in exon 8 of FGG gene gamma chain in this family.This mutation was first reported in China. 2.The ?Ala327Val heterozygous mutation in exon 8 of FGG gene leads to the disorder of the arrangement of fibrin monomer and the damage of D: D interaction,which disturbs the formation of ?-? dimer between fibrin monomers and leads to the serious damage of aggregation function.Part two: Evaluation of coagulation function in patients with congenital dysfibrinogenemia caused by ? Arg275 mutation by thromboelastographyBackground & objective : Thromboelastography(TEG)is a blood coagulation assay that simulates the internal environment of the human body in vitro by collecting whole blood samples,activating the coagulation system,and dynamically analyzing the whole process of blood clot formation and proteolysis(fibrinolysis).At present,TEG is rarely used in CD research,but the pathogenesis of CD is complex,CD patients have various clinical manifestations,and the routine blood coagulation function test for CD patients is relatively one-sided and single,while TEG can be used to evaluate the coagulation function of CD patients in an all-round way,and to diagnose the congenital dysfibrinogenemia.It is of great value in the diagnosis,differential diagnosis and evaluation of coagulation function of patients with CD.In this study,TEG was used to detect patients with CD caused by ?Arg275 mutation,to investigate the effect of this mutation on coagulation function in CD patients.Methods:In this study,we collected 24 patients with CD caused by ?Arg275 mutation and 84 normal controls,and detected their coagulation function with TEG to explore the effect of ?Arg275 mutation on coagulation function in patients with CD.At the same time,routine blood coagulation tests including APTT?PT?TT and Fg(Clauss method PT-derived method)were performed in patients with CD caused by ?Arg275 mutation;liver and kidney functions were also examined;DNA was extracted from peripheral blood cells of patients with CD,and the base sequence of the amplified fragment was detected;The aggregation of fibrinogen in plasma of CD patients was monitored dynamically to understand the function of fibrinogen aggregation in CD patients;The mutation sites of ? Arg275 were analyzed by amino acid model.Results:TEG test results showed that,compared with the control group,the K value of the experimental group was significantly longer(4.04±1.20 vs 2.06±0.46 min,P<0.05),and the Angle value was significantly lower(49.10±7.55 vs 60.76±8.75°,P<0.05),which indicated that the rate of clot aggregation was decreased and the function of fibrinogen was decreased in the experimental group.The routine examination of coagulation function showed that the plasma TT level of experimental group was significantly prolonged(31.83±4.33 s,reference range:9-15s),functional fibrinogen concentration(Clauss method)was significantly lower than fibrinogen antigen concentration(PT-derived method)(0.62 ± 0.17 vs 3.87±0.96g/L,P < 0.05,reference range: 2-5g/L).There was no abnormality of liver and kidney function in experimental group.Gene sequencing results showed that the mutation site of the experimental group was ? Arg275.The results of fibrinogen aggregation showed that the change of OD value of fibrinogen aggregation curve in the experimental group was smaller than that in the control group,and the maximum OD value(average 0.20Abs)was lower than that in the control group(0.32Abs),suggesting that the aggregation rate of fibrinogen in the experimental group was slower than that in the control group,and the aggregation of fibrinogen in the experimental group was defective.The modeling analysis shows that the mutation of ? Arg275 affects the functional region of the protein and the structure of the protein changes locally,which affects the stability of the structure.Conclusion:The type of fibrinogen function defect in CD patients was studied by thromboelastography.The results showed that the mutation of Arg at position 275 of gamma chain to His or Cys could lead to the damage of D: D interaction between fibrinogen molecules and cause aggregation disorder.The fibrinogen function in patients with CD was evaluated by thromboelastogram test,the results showed that the Arg mutation at position 275 of the gamma chain was capable of either His or Cys,resulting in aggregation dysfunction,possibly due to impaired D: D interaction between the fibrinogen molecules.