OBJECTIVETo investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia.

METHODSThe plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Thrombin fibrin aggregation curve were detected in the plasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed, and then transfected into COS-7 cells. The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA.

RESULTSAPTT, PT, TT were significantly longer in the proband. Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry, respectively. Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband. The proband's father, mother, brother and son were heterozygous. The polymerization curves of the patient did not show a lag phase or final turbidity, compared with the normal controls. Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions. It also could not detect the truncated Bβ chain under reducing conditions. Abnormal fibrinogen molecule (molecule weight>340 000) were found in transfected COS-7 cells by Western blot, which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absent in culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47 ± 0.30) μg/ml vs (2.65±0.60) μg/ml, P=0.0889]; However, the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ± 0.01) μg/ml vs (3.80±0.80) μg/ml, P=0.0001].

CONCLUSIONCongenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.

Afibrinogenemia ; congenital ; etiology ; genetics ; Fibrinogen ; genetics ; Frameshift Mutation ; Humans ; Male ; Mutagenesis, Insertional ; Pedigree ; Young Adult

OBJECTIVE: To analyze the phenotype and genotype of a pedigree affected with congenital dysfibrinogenemia. METHODS: Liver and kidney functions of the proband and her relatives were determined. Coagulation tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time(TT), fibrin(ogen) degradation products (FDPs), D-dimer(D-D) and the calibration experiment of protamine sulfate of against plasma TT were detected in the proband and her predigree members. The activity and antigen of fibrinogen (Fg) in plasma were measured by Clauss method and immunonephelometry method, respectively. All of the exons and exons-intron boundaries of the three fibrinogen genes (FGA, FGB and FGG) were subjected to PCR amplification and Sanger sequencing. Potential influence of the suspected mutations were analyzed with bioinformatics software including PolyPhen-2, SIFT and Mutation Taster. RESULTS: The proband had normal PT, APTT, FDPs, D-D and prolonged TT (31.8 s). The activity of fibrinogen (Fg) in plasma was significantly decreased but the antigen was normal. Genetic analysis revealed a heterozygous c.92G>A (p.Gly31Glu) mutation in exon 2 of the FGA gene. Family studies revealed that the mother carried the same mutation. Bioinformatic analysis suggested that the mutation may affect the function of Fg Protein. CONCLUSION The dysfibrinogenemia was probably caused by the novel Gly31Glu mutation of the FGA gene.
Afibrinogenemia ; congenital ; genetics ; DNA Mutational Analysis ; Female ; Fibrinogen ; genetics ; Humans ; Mutation ; Pedigree ; Phenotype

OBJECTIVE: To explore the coagulation deficit and genetic basis for a Chinese pedigree affected with Congenital dysfibrinogenemia (CD). METHODS: Peripheral venous blood samples of the proband and her family members (including 4 individuals from three generations) were subjected to routine blood test and assays of liver and kidney functions and viral hepatitis to exclude related diseases. Clauss method and DFg-PT method were used to determine the fibrinogen activity (Fg:C), and an immunoturbidimetric assay was used to determine the level of fibrinogen antigen (Fg:Ag). All of the exons (22 in total) and their flanking sequences of the FGA, FGB and FGG genes were amplified by PCR and directly sequenced. Variants in the coding regions of the three genes and transcriptional splicing sites were screened by using Mutation Surveyor^TM software. RESULTS: The Clauss method showed that Fg:C was significantly reduced in the proband and her father, whilst her mother and son were normal. With the DFg-PT method, the proband, her parents and son were all within the normal range. The Fg:C/Fg:Ag ratio of the proband and her father was lower than 0.7, whilst her mother and son were above 0.7. No significant change in the prothrombin time, activated partial thromboplastin clotting time and thrombin time was noted. Two genetic variants were detected, which included a homozygous missense variant in the FGA gene [c.991A>G (p.Thr331Ala)], which was predicted to be benign, and a heterozygous missense variant of the γ chain of the FGG gene [c.1211C>G (p.Ser404Phe)], which is located in a conserved region and unreported in the CLINVAR/HGMD/EXAC/1000G databases and literature. CONCLUSION This pedigree has conformed to the autosomal dominant inheritance of CD. The c.1211C>T (p.Ser404Phe) missense variant of the γ chain of the FGG gene probably underlay the pathogenesis of CD in this pedigree. The variant was unreported previously and named as "Fibrinogen Harbin II Ser404Phe".
Female ; Humans ; Afibrinogenemia/congenital* ; East Asian People ; Fibrinogen/genetics* ; Mothers ; Mutation ; Pedigree

OBJECTIVETo identify the genetic defect underlying congenital afibrinogenemia in a Chinese family.

METHODSPlasma fibrinogen (Fg) was assessed by both Clauss method and immunonephelometry. Genomic DNA was isolated from peripheral blood of the proband and 13 members of her family. All the exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Restriction endonuclease analysis was performed for the PCR products of the family members and 50 healthy donors to exclude gene polymorphism.

RESULTSNo Fg was detected in the plasma of the proband and her father by Clauss method, while low levels (< 0.02 g/L) were detected by immunonephelometry. A homozygous C to T mutation was found in the two cases at nucleotide 3108 in exon 4 of FGA gene, resulting in a null mutation which encoded severely truncated alpha-chains owing to its premature termination at the Gln 150 codon. The C-->T mutation eliminated a unique recognition site for restriction enzyme RsaI. The PCR amplified fragments of the proband and her father could not be digested by RsaI, showing that they are homozygous. Her mother and some family members are heterozygous at this site since the fragment could partly be digested, while the same fragment of controls could be completely digested as expected.

CONCLUSIONThe Gln (CAG)-->150stop (TAG) nonsense mutation in FGA gene is a novel genetic defect of congenital afibrinogenemia which, to our knowledge, has not been described before.

Adolescent ; Afibrinogenemia ; congenital ; genetics ; Base Sequence ; Codon, Nonsense ; DNA Mutational Analysis ; Exons ; genetics ; Female ; Fibrinogen ; genetics ; Humans ; Male ; Pedigree

OBJECTIVETo identify the fibrinogen (Fg) gene mutations in a Chinese pedigree of congenital afibrinogenemia.

METHODSThe plasma Fg activity and protein of the proband and his family members were detected. Genomic DNA was isolated from the peripheral blood mononuclear cells. All the exons and exon-intron boundaries of fibrinogen gene were amplified by PCR and sequenced thereafter.

RESULTSTwo mutations, 7972 del G in FGB and T2543A in FGG, were found in the proband.

CONCLUSIONSFGG2543 is a polymorphism site, which lead to the polymorphism of gamma144 I/K. The G deletion at base 7972 of FGB contributes to the frameshift mutation after amino acid 419, resulting in the truncated beta chain without the terminal 27 amino acids. The latter may contributes to the pathogenetic mechanisms in Chinese congenital afibrinogenemia patients. The G deletion at base 7972 of FGB is identified for the first time.

Adult ; Afibrinogenemia ; congenital ; genetics ; metabolism ; Base Sequence ; Blotting, Western ; DNA Mutational Analysis ; Exons ; genetics ; Female ; Fibrinogen ; genetics ; Humans ; Introns ; genetics ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction

OBJECTIVETo identify the genetic mutation underlying congenital hypofibrinogenamia in a Chinese pedigree.

METHODSStandard coagulation tests including the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), plasminogen activity (PLG:A), D-Dimer (DD) and fibrin degradation products (FDP) were tested with fresh plasma using a STA-R analyzer. The activity of fibrinogen (Fg:C) and fibrinogen antigen (Fg:Ag) were measured respectively with the Clauss method and immunoturbidimetry. All exons and exon-intron boundaries of the fibrinogen Aα-, Bβ-, and γ-chain genes (FGA, FGB and FGG) were amplified by PCR followed by direct sequencing. Suspected mutation was confirmed by reverse sequencing and analyzed with a Swiss-PdbViewer.

RESULTSThe PT level in the proband was normal, while the APTT and TT were slightly prolonged. The functional and antigen fibrinogen levels were both significantly reduced (0.91 g/L and 0.95 g/L, respectively). Similar abnormalities were also found in her father, elder sister, son and niece. The coagulant parameters of her mother were all within the normal range. Genetic analysis has reveled a heterozygous A>C change at nucleotide 5864 in exon 7 of γ gene in the proband, predicting a novel Lys232Thr mutation. The proband's father, elder sister, son and niece were all carriers of the same mutation. Protein model analysis indicated that the Lys232Thr mutation did not disrupt the native network of hydrogen bonds, but has changed the mutual electrostatic forces, resulting in increased instability of the protein.

CONCLUSIONThe heterozygous Lys232Thr mutation identified in the FGG gene probably underlies the hypofibrinogenemia in this pedigree.

Adult ; Afibrinogenemia ; congenital ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Female ; Fibrinogen ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Pedigree ; Peptide Fragments ; genetics ; Young Adult

OBJECTIVETo assess the platelet and plasma concentrations of fibronectin (Fn) and fibrinogen (Fg) in congenital fibrinogenopenic (FgP) patients and explore their role in inducing platelet adhesion and aggregation.

METHODSA FgP family was selected as study group and the platelets isolated and purified to assess concentrations of Fn and Fg in platelets, alpha-granules and plasma with Western blotting, immuofluoresence staining and flow cytometry (FACS), respectively, the expression of platelets GP II b/III a by FACS.

RESULTSThe concentration of platelets Fn in FgP patients is higher than that in controls, and is higher in homozygote than in heterozygote. In contrast, plasma Fn levels were identical in all samples. The amount of platelet Fg from FgP patients is lower than that from the controls and positively correlated with the concentration of their plasma Fg. No difference in the expression of platelet GP II b/III a had been found.

CONCLUSIONIt suggested that increased platelet Fn could partially compensate the lack of Fg and lead the platelet adhesion and aggregation.

Afibrinogenemia ; congenital ; metabolism ; pathology ; Blood Platelets ; metabolism ; pathology ; Cell Adhesion ; physiology ; Female ; Fibrinogen ; genetics ; metabolism ; Fibronectins ; blood ; genetics ; metabolism ; Heterozygote ; Homozygote ; Humans ; Male ; Pedigree ; Platelet Aggregation ; physiology ; Platelet Membrane Glycoproteins ; metabolism