The molecular basis of hypofibrinogenaemia and dysfibrinogenaemia
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Three novel fibrinogen variants were identified and characterised in conjunction with the further investigation of three cases with previously identified mutations. Fibrinogen Avon (p.BβGly290fsX308) was identified in a patient with hypofibrinogenaemia, with no clinical symptoms of a bleeding or thrombotic tendency. The mutation is predicted to cause read through into intron six, where a premature stop codon is encountered after the incorporation of 18 new residues. The translated Bβ chain would be 33% smaller than the normal Bβ chain, and would be missing most of the D domain, from the distal region of the snout onwards. Another novel Bβ chain variant, fibrinogen Mount Eden (Bβ440Trp→Stop), was identified in a hepatitis C positive patient with hypofibrinogenaemia, and a prolonged thrombin clotting time (TCT). The predicted translated chain would be missing the second strand of the five-stranded p-sheet, which is the major stmctural feature of the BβD domain. Mass spectrometry of isolated Bβ chains and tryptic digests, SDS-PAGE, and reverse phase HPLC revealed that neither the fibrinogen Avon or Mount Eden chains were incorporated into plasma fibrinogen, suggesting that both variant chains are unstable and are degraded intracellu1arly. Studies on the Mount Eden family revealed that the mutation did not segregate with the prolonged TCT, suggesting that this is probably secondary to the patient's hepatitis C infection. Fibrinogen Perth (p.AαPro495fsX518), was detected in a young woman with a history of menorrhagia and easy bruising. She had a prolonged TCT, indicative of dysfibrinogenaemia. Her gravimetric fibrinogen concentration was greater than the functional fibrinogen concentration. Fibrinogen Perth results from the deletion of a single cytosine (heterozygous) at nucleotide 4841 of the α gene, predicting a frame shift and the incorporation of 23 new amino acids before termination after residue 517. The truncated Aα chain was visualised by SDS-PAGE as a broadened 340 kDa band, and tryptic mapping of isolated Aα chains revealed that variant AαPerth chains were present in plasma at a low level of 0.14:0.86 (AαPerth/AαA ). This suggests that AαPerth chains may be out-competed by normal Au chains during molecular assembly, however, nonsense mediated decay of AαPerth mRNA cannot be ruled out. Fibrinogen Perth had abnormal polymerisation kinetics, particularly affecting lateral aggregation, and the resulting clots had greatly decreased permeability. Scanning electron microscopy (SEM) analysis revealed that fibrinogen Perth clots are composed of a dense network of fibres that are slightly thinner than normal, while fibrinolysis of fibrinogen Perth clots occurred at the same rate as the control. Another patient was found to be heterozygous for a 13 bp deletion of nucleotides 4783- 4795 in the α gene, translating to a frameshift and the incorporation of four new residues before premature termination after residue 479 (fibrinogen Lincoln). This represents the second identification of this mutation. However it is unknown whether this is an independent occurrence of the mutation, as genotyping of linkage markers to reveal the mutation's haplotype background in the two cases was unable to be performed. Like fibrinogen Perth, the tnmcated AuLincoln chain is expressed in plasma at the low level of 0.23:0.77 (AαLincoln:AαA). Polymerisation kinetics were abnormal, with the main defect also in lateral aggregation, while fibrinolysis was slightly faster than the control. Permeation studies revealed a decrease in permeability, indicative of a clot containing small pores. SEM analysis revealed a dense network of fibres that were thinner than the control. The fibrinogen Perth and Lincoln variants support the role of the AαC domain in lateral aggregation and the notion that an excess of Aα chains is present in the ER. The fibrinogen Dunedin variant (γ82Ala→Gly) was identified in a man with hypofibrinogenaemia, representing the third finding of this substitution. All three findings of the fibrinogen Dunedin mutation were identified on the same haplotype background, suggesting a founder effect for this mutation in New Zealand. The previous cases were complicated by the presence of other genetic variations, however, taken together the three findings of the same mutation in conjunction with low fibrinogen levels, suggest that it does cause hypofibrinogenaemia. However, the mechanism behind this is not known. The substitution occurs in a plasmin sensitive region in the coiled-coil, and may impair helix packing, causing increased extracellular degradation and thus hypofibrinogenaemia. Here, fibrinogenolysis and fibrinolysis experiments revealed that fibrinogen Dunedin is not degraded by plasmin any faster than normal fibrinogen, indicating that intracellular degradation is a more likely explanation of the hypofibrinogenaemia. The polymerisation kinetics and clot structure of a previously identified variant, fibrinogen Geraldine (BβArg14→Cys) were investigated during this study. This variant was originally discovered in a family with a history of severe thrombosis. Fibrinogen Geraldine has aberrant po1ymerisation kinetics, with a decreased V max and final turbidity, indicating defects with proto fibril formation and lateral aggregation. Permeation studies revealed a decrease in clot permeability, while SEM analysis showed a clot composed of mainly thick, loosely packed fibre aggregates surrounded by thinner fibres. The abnormal clot architecture is probably the cause of thrombosis in the effected patients. Fibrinolysis studies, although variable, revealed a significantly slower lysis rate for fibrinogen Geraldine, indicating that the abnormal clot structure may impair tPA and plasminogen binding or movement of these enzymes through the clot, causing the associated thrombotic disease. Several novel po1ymorphisms were also identified throughout this study; -1051 G/T; -946 TA repeat; -3G/A. The molecular basis of the Taq I polymorphism was also determined. The above polymorphisms were found to be in linkage disequilibrium with each other and the previously identified Rsa I polymorphism. Together these polymorphisms define a haplotype block which segregates with the α gene. Particular haplotypes within this block (E G and I) containing the A alleles for the -3 and Rsa I sites may be of particular interest to investigate in the future, as they are associated with elevated fibrinogen levels and thromboembolic disease respectively.