Aims A novel heterozygous variant, FGA c.169_180+2 del (designated fibrinogen Shanghai), was identified in a patient with dysfibrinogenemia with antiphospholipid antibody syndrome (APS) and recurrent venous thrombosis, and in his asymptomatic father. We aimed to reveal the functional implication of structural change caused by this variant. Methods Transcription analysis was performed with FGA minigene transfection assay to evaluate the impact of nucleosides deletion on mRNA editing. The fibrinogen isolated from propositus' plasma was used to characterise its functional defects. Fibrin polymerization and clot lysis experiments were performed by optical measurement of turbidity. Thrombin-catalysed fibrinopeptide release was analysed by the reversed-phase, high-performance liquid chromatography. The ultrastructures of fibrin clots were visualised by scanning electron microscopy. Results FGA c.169_180+2 del led to an aberrant mRNA with exon 2 skipping and encoded an shortened Aα chain with 42 amino acids truncation at its N-terminal. The propositus' fibrinogen had an impaired release of fibrinopeptide A and abnormal polymerization with a significantly prolonged lag time, a slower maximum slope and reduced final turbidity. The fibrin clot formed with propositus' fibrinogen showed thicker fibres with looser network structure. Clot lysis was normal using the purified fibrinogen but was significantly impaired using the plasma sample from propositus, compared with that from his father. Conclusions Fibrinogen Shanghai results in N-terminal truncation of Aα chain, which does not interfere with synthesis, assembly or secretion of fibrinogen, but compromises fibrin polymerization and clot formation. APS at least partially contributes to the development of thrombosis in the propositus.

INTRODUCTION:Congenital dysfibrinogenemia (CD) is a rare qualitative disorder of fibrinogen (Fg) with heterogeneous clinical manifestations. We aimed to analyze clinical phenotype and molecular basis of 102 Chinese CD patients and to evaluate the application of thromboelastography (TEG). MATERIALS AND METHODS:Clinical manifestations were recorded and quantified using the consensus ISTH bleeding assessment tool. Kaolin activated TEG and functional Fg TEG were applied in 30 patients. Genetic analysis of Fg genes were performed by direct sequencing. RESULTS:27.5% patients experienced bleeding, 3.9% had thrombosis and 68.6% were asymptomatic. Females were more prone to experience bleeding (P=0.01). Significant difference (P<0.05) in TEG results were found between patients with hot-spot mutations at AαArg35(16) and γArg301(275), but were not identified between patients with and without bleeding. Normal TEG results were found in patients with mutations at AαArg35(16), AαPro37(18) or AαArg38(19). Six novel mutations were identified, including AαGly33(14)del, AαAsp57(38)_Trp60(41)delIVS2+1_+2GTdel, AαPhe742(723)Tyr, γAsn334(308)Thr, γGly335(309)Cys and γTrp395(369)Leu. CONCLUSIONS:CD patients have similar clinical manifestations and hot-spot mutations worldwide with no ethnic difference. TEG results could not indicate the bleeding risk in patients, but priority of mutation screening at thrombin cleavage site or polymerization site on Aа chain may be given if TEG results are normal.

We report a rare case of congenital hypodysfibrinogenaemia due to maternal uniparental disomy of chromosome 4 (mat UPD 4) and a maternally inherited novel nonsense mutation Trp323X in the fibrinogen Bβ chain (FGB) gene. Western blot analysis of patient's plasma revealed an abnormal fibrinogen which consisted of truncated Bβ chain and normal Aα and γ chains. Patient's clinical history and laboratory evidence are presented. Microsatellite genotyping analysis revealed a mixed nature of heterodisomy and isodisomy along chromosome 4. High density SNP genotyping array analysis further confirmed the mat UPD 4 and defined two segments of chromosome 4 (4pter-p15.33 and 4q31.21-4q32.3) as maternal isodisomy (iUPD4) and the remaining regions as maternal heterodisomy (hUPD4), with the FGB gene carrying the mutation resided in the iUPD4 region on the long (q) arm. It was predicted that the segmental nature of iUPD and hUPD was caused by three recombination events at positions around 167.96 cM, 145.51 cM and 14.40 cM on chromosome 4 followed by a meiosis I non-disjunction. This case is clinically and molecularly unique and offers an opportunity for understanding novel mechanisms of congenital hypodysfibrinogenaemia associated with complex UPD and fibrinogen secretion.

Objective To analyze the phenotype and genotype of a Chinese family with inherited hypofibrinogenemia,and to investigate its molecular mechanism.Methods Peripheral blood was collected from seven people of this family and then plasma was separated.Activated partial thromboplastin time ( APTT),prothrombin time ( PT),thrombin time ( TT),reptilase time ( RT),the activities of antithrombin( AT∶ A ),protein C ( PC ∶ A ) and protein S ( PS ∶ A ) were tested.The activity and antigen of plasma fibrinogen were analyzed by Clauss method and immunoturbidimetry method,respectively.The fibrinogen peptide chain of the proband was semiquantitatively assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Thrombin generation test was performed by calibrated automated thromhogram.The dynamic process of blood coagulation was evaluated by the thrombelastography (TEG).Genomic DNA was extracted from the peripheral blood.The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes FGA,FGB and FGG were amplified by polymerase chain reaction ( PCR ) and analyzed by direct sequen(c)ing.Results The activity and the antigen levels of the proband' s plasma fibrinogen were reduced to 0.48 g/L and 0.68 g/L,respectively.TT prolonged to 29.2 s and RT prolonged to 75.8 s.The assays of SDS-PAGE showed no abnormal molecular weight of fibrinogen.Peak height of thrombin generation was reduced to 249.93 nmol/L and endogenous thrombin potential was reduced to 1007.0 nmol · L-1 · min.Hypocoagulability state of the whole blood was found by TEG test.The coagulation index was - 8.6.The proband was diagnosed as inherited hypofibrinogenemia by phenotype analysis.Two mutations (Gln143Pro and g.4642delC) were found in the proband's fibrinogen Aa-chain gene,Gln143Pro came from her mother and g.4642delC came form her father.Conclusion Compound Heterozygous Mutations (Gln143Pro and g.4642delC ) of fibrinogen Aa-chain causes the proband congenital hypofibrinogenemia.

Objective To investigate the relationship between phenotypes and genotypes in three Chinese pedigrees with inherited antithrombin deficiency.Methods The routine coagulation tests including activated partial thromboplastin time(APTT),prothrombin time(PT),fibrinogen(Fg) and thrombin time(TT) were performed.The activities of protein C(PC:A),protein S(PS:A),and antithrombin(AT:A) were tested with chromogenic substrate assay.The antigen of AT(AT:Ag) was tested with immunoturbidimetry methods.The molecular weight(MW) and amount of AT was assessed by Western blot.The sequences of all the exons and exon-intron boundaries of the AT gene were amplified by PCR and analyzed by direct sequencing.The novel mutation in AT gene was tested in 100 normal persons to exclude polymorphism.The heterozygous deletion was checked and identified by sequencing the PCR products constructed in TA cloning vector.Results Three probands had recurrent venous thrombosis.The levels of APTT,PT,Fg,TT,PC:A and PS:A were normal,AT:A in the plasma was 60%,52% and 60% of the normal levels in each of the 3 probands,with respective AT:Ag of 16.9 mg/dL,14.1 mg/dL and 11.4 mg/dL.Western blot showed that their AT MW was normal(58 kD) but the antigen levels were lower than normal.Three heterozygous mutations of Leu340Phe,Phe122del and Phe123Cys were identified in the 3 probands.In each of the 3 pedigrees,the same genotypes(heterozygous mutations) were identified in the family members with similar low level of AT as the probands.However,only the proband′s father in pedigree 2 had venous thrombosis once,there were no other family members having had thrombosis in both pedigree 1 and 3.Conclusion The three probands with inherited antithrombin deficiency were each caused by mutations of Leu340Phe,Phe122del and Phe123Cys.Both Leu340Phe and Phe123Cys were internationally first reported.

OBJECTIVE To analyze the phenotype and genotype in three Chinese pedigrees with inherited dysfibrinogenemia. METHODS Laboratory tests including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), and the activities of antithrombin (AT:C), protein C (PC:C) and protein S(PS:C) were detected in three pedigrees. The activity and antigen of plasma fibrinogen (Fg) were analyzed by Clauss and immunoturbidimetry methods, respectively. The Fg of three probands was assessed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of all the exons and exon-intron boundaries of the three Fg genes FGA, GFB and FGG were amplified by PCR and analyzed by direct sequencing. RESULTS Three probands had normal APTT, PT, PC:C, PS:C and AT:C, but prolonged TT and RT. The activity levels of the 3 probands's plasma Fg were reduced, but antigen levels were normal. Western blot and SDS-PAGE showed no abnormal molecular weight of Fg. The 3 heterozygous mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys were identified in the 3 probands, respectively. CONCLUSION The three probands with dysfibrinogenemia were caused by the mutations of γ Arg275His, Aα Pro18Leu and Aα Arg16Cys, respectively. Both Aα Pro18Leu and Aα Arg16Cys were first reported in Chinese population.