The ß-chain mutation p.Trp433Stop impairs fibrinogen secretion: A novel nonsense mutation associated with hypofibrinogenemia.

BACKGROUND: Congenital hypofibrinogenemia is characterized by proportional decreases in fibrinogen activity and immunoreactive fibrinogen levels. Here, we describe a new case with the bleeding risk identified in our hospital. METHODS: The proband was cut and bled for 3 h. Coagulation testing, gene analysis, thrombelastogram, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in vitro plasmid construction, and functional analyses were performed to explore the pathogenic mechanism. RESULTS: Coagulation testing of the male proband revealed low levels of fibrinogen detected by two methods (the Clauss method and the PT-derived method); his two sons had normal coagulation results. DNA sequencing of the proband revealed a heterozygous point mutation in exon 8 of the FGB gene causing Trp→Stop substitution and a polymorphic site (p.Leu92Phe). Human Trp433 was found to be highly conserved. SDS-PAGE showed that the fibrinogen level of the proband was markedly lower than that of healthy controls. Using high-performance liquid chromatography-mass spectrometry, a mutated Bß chain was not detected in circulation. In vitro expression analyses indicated that the mutation affected the secretion of fibrinogen. The TEG results indicated that the proband had a prolonged K time, a lower CI value, and a lower angle value. CONCLUSION: We report a new case with a novel nonsense mutation that resulted in hypofibrinogenemia. The results indicate that the nonsense mutation may cause misfolding of the D domain, which then affects the secretion of fibrinogen.

Apolipoprotein A-IV as a novel gene associated with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder, affecting 6-10% of women of reproductive age. The etiology remains poorly understood. To investigate the differentially expressed proteins from PCOS patients versus healthy women, the protein expression in follicular fluid was analyzed using two-dimensional electrophoresis (2-DE). Since follicular fluid contains a number of secretory proteins required for oocyte fertilization and follicle maturation, it is possible that follicular fluid can be used as a provisional source for identifying pivotal proteins associated with PCOS. In this study, six overexpressed proteins [kininogen 1, cytokeratin 9, antithrombin, fibrinogen γ-chain, apolipoprotein A-IV (apoA-IV) precursor and α-1-B-glycoprotein (A1BG)] in follicular fluids from PCOS patients were identified with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and nano LC-MS/MS. Western blot analysis confirmed that the protein expression levels of apoA-IV precursor and A1BG were increased in follicular fluid from PCOS patients compared with those from normal controls. The analysis of protein expression for other proteins revealed individual variation. These results facilitate the understanding of the molecular mechanisms of PCOS and provide candidate biomarkers for the development of diagnostic and therapeutic tools.

Novel Aalpha chain truncation (fibrinogen Perth) resulting in low expression and impaired fibrinogen polymerization.

A young woman with a history of menorrhagia and easy bruising presented with a functional fibrinogen concentration of 1.8 mg mL(-1), a gravimetric concentration of 3.3 mg mL(-1) and a prolonged thrombin clotting time of 32 s. Both reverse phase analysis and reducing SDS-PAGE revealed a normal profile of Aalpha, Bbeta, and gamma chains. However, non-reducing gels revealed a broadened 340-kDa band, while the 305-kDa band was normal, suggesting a C-terminal truncation of the Aalpha chain. DNA sequencing of all exons and intron boundaries revealed a single heterozygous cytosine deletion at nucleotide 4841 of the Aalpha gene predicting a frameshift and the incorporation of 23 new residues (LMKLPSSTLPQLEKHSQVSSHLC) before termination after residue 517. In agreement with a predicted mass decrease of 9953 Da, the measured mass of the Aalpha(Perth) chain was 56 242 Da, while that of the normal Aalpha(A) chain was 66 189 Da. Tryptic mapping of isolated Aalpha chains revealed a new [M + 2H] ion at 607 m z(-1), corresponding to the predicted penultimate peptide LPSSTLPQLEK. The variant chain was poorly incorporated into plasma fibrinogen at a ratio of Aalpha(Perth)/Aalpha(A) of 0.15 : 1, suggesting the Aalpha(Perth) chain might be out-competed by normal chains during molecular assembly in the hepatocyte. Despite the low expression, polymerization curves showed a decreased V(max) and final turbidity, suggesting the fibrinogen Perth clots are composed of thinner fibers. However, the fibrinolytic rate was very similar to that of the control.

The impaired polymerization of fibrinogen Longmont (Bbeta166Arg-->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers.

This study identified a new substitution in the Bbeta chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site "a." Thus, the sites "A" and "a" can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unable to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg166-->Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils. (Blood. 2001;98:661-666)

Detection of a complex that associates with the Bbeta fibrinogen G-455-A polymorphism.

The promoter region of the Bbeta fibrinogen gene containing the polymorphic site (G-455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different -455 nucleotide substitutions of the Bbeta promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position -455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at -455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bbeta gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele.

Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val --> Asp).

A novel mechanism of molecular disease was uncovered in a patient with prolonged thrombin time and a mild bleeding tendency. DNA sequencing of the fibrinogen A alpha chain indicated heterozygosity for a mutation of 20 Val --> Asp. The molar ratio of fibrinopeptide A to B released by thrombin was substantially reduced at 0.64 suggesting either impaired cleavage or that the majority of the variant alpha chains lacked the A peptide. The latter novel proposal arises from the observation that the mutation changes the normal 16R G P R V20 sequence to R G P R D creating a potential furin cleavage site at Arg 19. Synthetic peptides incorporating both sequences were tested as substrates for both thrombin and furin. There was no substantial difference in the thrombin catalyzed cleavage. However, the variant peptide, but not the normal, was rapidly cleaved at Arg 19 by furin. Predictably intracellular cleavage of the Aalpha-chain at Arg 19 would remove fibrinopeptide A together with the G P R polymerisation site. This was confirmed by sequence analysis of fibrinogen Aalpha chains after isolation by SDS-PAGE. The expected normal sequence was detected together with a new sequence (D V E R H Q S A-) commencing at residue 20. Truncation was further verified by nonreducing SDS-PAGE of the NH2-terminal disulfide knot which indicated the presence of aberrant homo- and heterodimers.