Study On The Pathogenesis Of Congenital Fibrinogen Disorders

Background & objectiveFibrinogen(Fg)is a plasma glycoprotein composed of 2964 amino acids,with a molecular weight of 340 kDa.Fg,also known as coagulation factor Ⅰ,can be converted into insoluble fibrin clots under the action of thrombin,and can mediate platelet aggregation and adhesion,which plays an important role in the process of anticoagulation.It is a symmetric dimer composed of Aα,Bβ and γchains,and Aα,Bβ and γ chains are encoded by FGA gene,FGB gene and FGG gene respectively.When the Fg(FGA / FGB / FGG)gene is defective,it can cause the abnormal Fg content and / or function,which leads to the congenital fibrinogen disorders(CFDs).CFDs can be divided into two types: one is the insufficient or complete lack of Fg synthesis,which is manifested by the decrease of Fg content in plasma,including congenital afibrinogenemia and congenital hypofibrinogenemia;the other is the defect of Fg structure and function,namely the abnormality of Fg quality,including congenital hypodysfibrinogenemia and congenital dysfibrinogenemia(CD).The clinical manifestations of CFDs are various: no obvious clinical symptoms,bleeding,thrombosis,etc.;some female patients may have menorrhagia,placental abruption during pregnancy,spontaneous abortion,postpartum hemorrhage,etc.Studies have found that even in asymptomatic CFDs patients,the risk of bleeding or thrombosis is still relatively high.The genetic pattern and mutation type of CFDs are diverse,but different mutation types and mutation sites cause different pathogenesis.Therefore,although the research on CFDs at home and abroad has been extended to the molecular level,the specific pathogenesis of CFDs has not been fully understood.In this study,we collected three families of CFDs,analyzed their clinical manifestations,related medical history and family history,combined with the results of routine coagulation function and liver and kidney function examination,detected gene mutation sites,Fg function analysis,amino acid conservation analysis and in vitro function test,and explored the pathogenesis of CFDs.MethodsThree CFDs families were collected in this study.(1)Three CFDs families were inquired about their history,investigated their families,and drew their genealogies;(2)Peripheral blood of the proband and its family members was extracted for routine coagulation function examination(PT,APTT,TT,Fg-Clauss method and Fg-PT algorithm)and liver and kidney function examination;(3)Blood DNA was extracted,and primers for all exons and flanking sequences of FGA,FGB and FGG genes were designed and synthesized.PCR amplification and product sequencing were carried out.The standard sequences of related genes in NCBI database were compared,and the specific mutation sites of patients’ genes were found and identified;(4)The plasma Fg was extracted,and the type of Fg defect was analyzed by SDS-PAGE electrophoresis and staining;(5)The presence of mutant peptide was detected by HPLC-MS/MS;(6)Detect Fg aggregation function;(7)Observation of fibrin clot structure by SEM;(8)Analysis of amino acid conservation of mutation site by Clustal X2.1 software;(9)The expression vectors of Fg wild type and mutant type were constructed and transfected into CHO cells to analyze the effect of gene mutation on Fg synthesis and secretion;(10)The whole coagulation state was evaluated by thromboelastography.Results(1)All the three proband patients had obvious clinical manifestations,such as bleeding without obvious incentive,intraoperative massive bleeding,postoperative bleeding,etc.The results of Fg-Clauss of proband Ⅰ and proband Ⅱ were significantly lower than those of Fg-PT algorithm;the consistency of Fg-Clauss and Fg-PT algorithm of proband Ⅲ were significantly reduced.There was no abnormality in the liver and kidney function of the four probands.(2)Sequencing of genes revealed that c.103 C>T heterozygous mutation(p.Arg35 Cys missense mutation)occurred in exon 2 of FGA and c.911 A>G heterozygous mutation(p.Thr331 Ala missense mutation)in exon 5 of FGA in proband Ⅰ;c.104 G>A heterozygous mutation(p.Arg35 His missense mutation)occurred in exon 2 of FGA of proband Ⅱ;c.1299 G>A heterozygous mutation(p.Trp433 Stop nonsense mutation)occurred in exon 8 of FGB and c.274C>T heterozygous mutation(p.Leu92 Phe missense mutation)occurred in exon 3 of FGG of proband Ⅲ.(3)Non-reducing SDS-PAGE and Coomassie brilliant blue staining showed that Fg polymer bands were found in three probands and normal controls;reducing SDS-PAGE and Coomassie brilliant blue staining showed that Aα,Bβ and γ bands were found in three probands and normal controls,and no abnormal bands were found;(4)HPLC-MS/MS showed that there were normal peptide and abnormal peptide in the plasma Fg of proband Ⅰand proband Ⅱ,but no mutant peptide was detected in the plasma Fg of probandⅢ;(5)Fg aggregation function showed that compared with the normal control group,the OD value of Fg aggregation curve of proband Ⅰ and proband Ⅱ had little change,obvious delay stagnation period,and the maximum OD value was low;Fg aggregation curve of proband Ⅲ had no obvious delay stagnation period,and the OD value change and the maximum OD value were not significantly different from the normal control group;(6)Scanning electron microscopy showed that the fibers of the proband Ⅰ were uniform in thickness and arrangement,the spatial structure of the fiber network was loose,and there were many fiber nodes;the fibers of the proband Ⅱ were uneven in thickness and arrangement,the spatial structure of the fiber network was loose and the pore diameter was increased,there were many fiber nodes,and the ends of the fibers were curled.the fibers of the proband Ⅲ were uniform in thickness and arrangement,the spatial structure of the fiber network was relatively loose and the pore diameter was slightly large,and the fiber nodes were rare.(7)The comparative analysis of amino acid conservation showed that Aα Arg35 and BβTrp433 were highly conserved.(8)In vitro functional test showed that the mutation of Aα Arg35 Cys and Aα Arg35 His did not affect the transcription and secretion of Fg,the mutation of Bβ Trp433 Stop may affect the normal secretion of Fg;(9)Thrombelastography showed that the R-value of the proband Ⅰdecreased;the result of the proband Ⅱ was in the normal range;the K-value of the proband Ⅲ increased,Angle value decreased and CI-value decreased.Conclusion1.Phenotypes and genotypes of four probands and their families were studied.Both proband Ⅰ and proband Ⅱ were diagnosed as hereditary abnormal fibrinogenemia.The proband Ⅲ were diagnosed as hereditary hypoproteinemia.2.The mutations of the proband Ⅰ(Aα Arg35Cys)and the proband Ⅱ(AαArg35His)of this study are mutation hot spots,which are consistent with other literature reports c.1299G>A heterozygous mutation(p.Trp433Stop)in exon 8 of FGB in the proband Ⅲ were found for the first time at home and abroad.3.The mutations of the proband Ⅰ(Aα Arg35Cys)and the proband Ⅱ(AαArg35His)can lead to the abnormal aggregation function.The mutation of the proband Ⅲ(Bβ Trp433Stop)may affect the normal secretion of Fg.4.The R value of thrombelastography in the proband Ⅰ decreased,indicating that the activity of factors in the body of the patient increased and the blood was in hypercoagulable state;the results of thrombelastography in the proband Ⅱ were all in the normal range,indicating that the activity of factors in the body of the patient and the coagulation state were normal;the K value in the results of thrombelastography in the proband Ⅲ increased,and the angle value decreased,indicating that the rate of aggregation of blood clots decreased,Fg function was low;the CI value decreased Low,indicating that the patient is in a low coagulation state,suggesting that it may have a higher risk of bleeding.