Genetic Diagnosis And Phenotype Analysis Of Hereditary FⅦ Deficiency And Identification,Structure And Function Analysis Of Fibrinogen γ His340Arg Mutant

Objective:To analyze the types of gene mutation and clinical characteristics in 12 patients with hereditary coagulation factor Ⅶ deficiency and investigate the structure and function for fibrinogen γHis340Arg mutant of combined congenital hypodysfibrinogenemia with factor XI deficiency in a Chinese family by gene analysis and pedigree survey.Methods:PT,APTT,Fg,TT,FⅦ:C and FⅪ:C were performed by one-stage assays.Clauss method and PT-derived method were used to detect fibrinogen activity and antigen.Genomic DNA was extracted from the peripheral blood of patients and their family members.The all exons and exon-intron of F7,F11 gene as well as fibrinogen genes(FGA,FGB and FGG)were amplified by PCR and analyzed by direct sequencing.Cluster X and Swiss-PdbViewer4.10 software were used to analyze the conservative and molecular modeling of fibrinogen γHis340Arg mutant.Using thrombin method to dynamically monitor the formation of fibrin polymer to analyze the function of mutant fibrinogen.Results:1.Six missense mutations were found in 12 patients.The p.Met366 Val and p.Cys418 Gly were reported for the first time.The p.Thr241 Asn was identified in 5 of 7 patients with F7 gene homozygous mutations,and the activity of FⅦ were 3.6%,4.1%,3.7%,3.5% and 4.0%,respectively.Clinical bleeding phenotypes were different among of them.The remaining two patients carried p.His408 Gln and p.Thr419 Met homozygous mutation.and the activity of FⅦ were 4.4% and 3.9%,respectively.The former had no tendency to spontaneous bleeding while the latter showed recurrent mild to moderate bleeding of the skin mucous membrane.Another four cases carried compound heterozygous mutations: p.Thr241 Asn and p.His408Gln(FⅦ:C :3.6 %),p.Thr241 Asn and p.Met366Val(FⅦ:C:3.6%),p.Thr241 Asn and p.Cys389Gly(FⅦ:C :2.5 %),p.Thr241 Asn and p.Cys418Gly(FⅦ:C :3.5 %).The disorder was manifested with a wide array of clinical bleeding events.Another patient only was found p.Thr241 Asn heterozygous mutation and its FⅦ:C was 2.7%,with no bleeding tendency.2.The fibrinogen activity and antigen of proband,grandmother,father,aunt as well as sister were measured to be respectively 0.33g/L and 1.77g/L,0.32g/L and 1.69 g/L,0.29g/L and 1.36 g/L,0.31g/L and 1.69 g/L,0.36g/L and 1.66 g/L;c.1097A>G in exon 8 of FGG was detected in the pedigree,which caused γ His340 Arg mutation.The proband’s grandfather fibrinogen activity was 2.93g/L,but the FⅪ activity decreased to 17.6%.FXI deficiency was a compound heterozygote inherited with missense mutations of c.434A>G in exon 5 as well as c.1253G>T in exon 11,which caused HGV p.His145 Arg and Gly400 Val mutations,respectively.The proband,his father and aunt had c.434A> G in exon 5 of F11 gene,his mother had no fibrinogen or F11 gene mutations.3.The 340 th histidine residue of fibrinogen γ chain protein is highly conserved among different species.Molecular modeling showed that the γ His340 Arg mutation does not affect the spatial structure of γ chain protein.Functional analysis revealed that the fibrin polymerization ability of patients with the γ His340 Arg mutation decreased.Conclusions:1.six missense mutations were found in 12 patients with hereditary FⅦ deficiency,two of which were reported for the first time,p.Thr241 Asn mutation may be a hot spot mutation site.2.There is no obvious correlation between FⅦ: C and clinical phenotype.3.The heterozygous mutation of exon 8 c.1097A>G(γ His340Arg)in FGG gene is a new mutant,which may cause abnormal fibrinogen function by interfering with the delay of fibrin polymer.4.Double heterozygous mutations of exon 5 c.434A>G(HGV p.His145Arg)and exon 11 c.1253G>T(p.Gly400Val)in F11 gene cause hereditary FXI deficiency in this family.