Diagnosis And Molecular Pathogenesis Of FGA Gene In Congenital Dysfibrinogenemia

Background and ObjectiveCongenital dysfibrinogenemia(CD)is a qualitative fibrinogen disorder caused by an abnormal fibrinogen molecule structure,leading to blood coagulation dysfunction.The clinical phenotype is highly heterogeneous,from no manifestations to bleeding and/or thrombotic events.A follow-up study has shown that asymptomatic propositi and relatives with the predisposing genotype are at risk to develop adverse outcomes during the natural course of the disease.The diagnosis of congenital dysfibrinogenemia is generally based on the laboratory examination,which shows discrepancy between fibrinogen activity detected by Clauss method and antigen levels detected by PT-derived method or immunoturbidimetry,and thrombin time(TT)was distinctly prolonged.It is important to identify CD gene mutation and to further explore the molecular pathogenesis of CD and to evaluate the risk of bleeding and thrombosis of CD patients.Methods(1)We collected 4 CD probands of their histroy data.(2)The whole genomic DNA of blood cells was extracted.Specific primers of fibrinogen geneswere designed and synthesised.All the exons and flanking sequences of FGA,FGB and FGG genes were amplified by PCR to obtain the target product.Direct sequencing analysis was performed to find the type of gene mutation.(3)We purified the plasma fibrinogen of the patient.Mass spectrometry was used to detect variant peptides.(4)In order to clarify the fibrinogen defect type,western blot was used to analyze the relative expression of fibrinogen peptide chains.(5)We performed kinetic measurement of fibrinopeptide release,fibrinolysis assays and fibrin polymerization curves measurement.(6)Scanning electron microscopy(SEM)was used to observe the fibrin network structure.Through those methods,the molecular pathogenesis of CD patients was discussed.Results(1) The results of coagulation test were as follows: fibrinogen antigen concentration detected by PT-derived method was normal,fibrinogen activity level detected by Clauss method was low,TT was significantly prolonged.?DNA sequencing of Proband ?,?and ? revealed a heterozygous point mutation in exon 2 of the FGA gene at the position g.1232G>A,which causes the substitution of A? 16 Arg to His.Proband ? had a heterozygous point mutation in exon 2 of the FGA gene at the position g.1233C>T,which causes the substitution of A? 16 Arg to Cys.?Mass spectrometry showed that fibrinogen mutation chains appeared in patients' circulation.(4)Western blot did not reveal any detectable differences in the electrophoretic mobility between the probands and healthy controls.(5)The amount and release rate of fibrinopeptide A of probands are damaged.Fibrin polymerization of probands showed a remarkablly prolonged lag phase,and the maximum OD was lower than that of the healthy control.In the clot lysis experiment,there was a significant prolonged stagnation period in the dissolution curve,with a 50% dissolution time delay.(6)SEMshowed that fibrin clots were formed by narrower fibrils,and that fibrin had many small branched fibers with twisted ends and large pores compared with healthy control.Conclution1.The mutations(A?16Arg?His and A?16Arg?Cys)we reported in this study were two mutation hotspots.2.In this study,we successfully studied the type of fibrinogen deficiency in CD patients by Western Blot.Combine with SDS-PAGE,Western blot did not reveal any detectable differences in the electrophoretic mobility between the probands and healthy controls.3.No matter what the substitution of A? 16 Arg to Cys or His,they all can caused thrombin-fibrinogen interaction abnormal,resulting in reduced release of fibrinopeptides,and further cause aggregation dysfunction and fibrinolytic resistance.That is the molecular pathogenesis of four families with congenital dysfibrinogenemia.