Identify Pathogenic Gene Of Fibrinogen Fga And Fgg Genes And Phenotypic Study Of Congenital Dysfibrinogenemia

Background and ObjectiveCongenital dysfibrinogenemia(CD)is a genetic disease that causes abnormal fibrinogen structure and function due to mutations in fibrinogen-encoding genes.The disease is usually autosomal dominant inheritance or codominant inheritance,and the minority is autosomal recessive.The clinical manifestation is complex,most patients do not have any clinical manifestations,while some patients suffer from thrombosis or hemorrhage,and very few patients have bleeding and thrombosis together,some female patients suffer from menorrhagia,recurrent miscarriage,and placental abruption.For the most patients without any clinical manifestations mainly identified accidentally by coagulation assay preoperative or physical examination,the typical laboratory test results revealed prolong thrombin time(TT),the level of fibrinogen activity(Clauss method)was significantly reduced while the antigen concentration was within the normal range.However,there are also very few patients whose laboratory test results are inconsistent with the above description.A few patients present with a normal or short TT also have been reported.Therefore,identify CDs via laboratory tests and gene analysis is necessary,which provide information sources for prenatal diagnosis and prepotency,and determine the prognosis and guide treatment by clinical phenotype analysis.Methods(1)We collect the blood specimens and history data for CD families and then draw the pedigree.(2)The whole genomic DNA was extracted from peripheral blood leukocyte,specific primers were designed and synthesized,then amplified all exons and their flanking sequences of fibrinogen genes via PCR,the nucleotide sequence of the amplified target fragment was detected by a sequencer and compare with NCBI's standard sequence in the gene database to find the mutation in the proband,and then the other members of the family were screened for relevant gene mutations.(3)Plasma fibrinogen from the proband and their family members were extracted and purified,and the relative molecular weight and relative expression of fibrinogen peptides were analyzed by Western blot;mutant peptides in plasma were analyzed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry.(4)Monitored the dynamic aggregation of plasma fibrinogen from the proband and normal control,in order to understand the aggregation function of fibrinogen from CD patients.(5)The network structure and fiber diameter in the fibrin clot were observed by scanning electron microscope.(6)Thrombelastograph was used to evaluate the coagulation function and predict the risk of hemorrhage or thrombosis of the probands.(7)The clinical manifestations of probands and their family members were analyzed and compaired with the other probans with the same mutation sites in the past 10 years.Results(1)By analyzed with gene mutation,the proband I,III and IV had C.902G>A missense mutation in exon 8 of the FGG gene that resulted in amino acid substitution 275 Arg?His in the ? chain(lacking signal peptide);the proband ? had C.104G>A missense mutation in exon 2 of the FGA gene that resulted in amino acid substitution 16 Arg?His in the A? chain(lacking signal peptide).(2)Western blot analysis of the fibrinogen A ? chain or ? chain did not reveal any detectable differences in the mobility and relative expression between the probands and the normal control;Analyzed by mass spectrometry,fibrinogen of four probands were found to contain both normal and mutated peptides,consistent with heterozygous mutations.(3)The fibrin polymerization of four probands changed significantly,the lag period was prolonged,and the maximum OD value was lower than that of normal control.(4)The fibrin clot structure was observed by scanning electron microscopy,the network structure of the proband ??? and ? were loose,with larger pore size,and the clots were comprised of uneven thickness fibrin fibers and arranged irregularly,the network structure of the proband II was denser with smaller pore size,the clot was comprised of uniform thickness fiber,and fiber nodes are more than normal control.(5)The thromboelastogram showed that the angles of proband I and III decreased,the corresponding k value increased,and the MA decreased,whereas the results of proband II and IV were within the normal range.(6)Four probands and family members with the same mutation sites did not have any clinical manifestations.Conclusions(1)Genetic analysis demonstrated that Arg275 His mutations occurred in the probands ??? and ?,Arg16 His mutations occurred in the proband ?,and all were fibrinogen mutation hotspots,consistent with other reports.(2)The thromboelastogram showed that the Angle values of the probands ? and ? decreased,the corresponding k value increased,and the MA decreased,indicating that the fibrinogen function was decreased,but the remaining normal fibrinogen still had the function of hemostasis;The thrombelastogram results for probans?and?were within normal ranges.None of the four probans had any risk of bleeding or thrombosis.