Fibrinogen Nottingham II: A novel Bβ Arg264gly substitution causing hypofibrinogenaemia

Thromb Haemost 2006; 96: 378–80 Plasma fibrinogen circulates as a 340 kDa glycoprotein made up of two identical subunits, each consisting of three different polypeptides (Aα, Bβ and γ chains). Its molecular structure consists of a central domain (E), containing the N-terminus of each chain, linked by a coiled coil region to two lateral domains (D) that contain the C-termini of the Bβ and γ chains (1). Hereditary fibrinogen disorders vary considerably in their underlying genetic defects and in their phenotypic and clinical consequences (2). The genetic and molecular study of these disorders have provided a better understanding of the regions of the fibrinogen molecule that are important in terms of function and structure (3). This study outlines the identification of a novel mutation in the fibrinogen BβD domain in a family with hypofibrinogenaemia, and the investigation of the significance of this mutation at the molecular level. The index case was 43-year-old asymptomatic woman with a reduced fibrinogen Clauss (1.2 g/l) and antigenic (1.0 g/l) fibrinogen concentration (normal 2.0–4.0), and a total clot weight of 1.2 g/l (normal 1.8–4.0). There was a family history of hypofibrinogenemia, which was diagnosed in her mother and both sisters. The only bleeding problem experienced in this family was postpartum hemorrhage, which occurred in all four female members with hypofibrinogenemia. However, the index case had fibrinogen levels within the normal range by the end of pregnancy (but considered low for full term pregnancy) and in her case postpartum hemorrhage was associated with atonic uterus. Genetic analysis of the index case by denaturing HPLC screening and nucleotide sequencing (4, primers available on request) revealed a heterozygous novel candidate mutation. This was a single base change in exon 6 of the FGB gene (c.882 A>G) leading to a codon change (AGG to GGG) predicted to result in the amino acid substitution Bβ Arg264Gly. This was confirmed in both sisters (DNA was not available from her mother) and was not detected in a screen of 60 control individuals using denaturing HPLC Reducing SDS-PAGE of purified plasma fibrinogen identified a normal pattern of Aα, Bβ and γ chains, and analysis by reverse phase HPLC (5) showed normal retention times and areas for all three peaks. However, mass spectrometry provided compelling evidence that the Bβ Arg264Gly mutation was the direct cause of hypofibrinogenemia Direct mass measurements of isolated Bβ chains from three controls gave an average mass of 54,212 ± 4 Da for the major monosialylated isoform, while the measured mass of the Nottingham chain was 54,215 Da. This strongly suggested that there was little or no expression of this variant Bβ chain in plasma fibrinogen; if present and fully expressed, the arginine to glycine mutation should cause a 99/2 Da decrease in overall mass. To confirm these findings, peptide mapping was performed (6). In addition to being heterozygous for Bβ Arg264Gly, the index case was also found to be heterozygous for the Bβ Arg448Lys polymorphism. Mass maps were therefore compared directly to a control sample with the same Bβ448 polymorphism (Fig. 1A). Non expression of the variant chain was confirmed by examination of the M+4H ion of peptide D-28–29 containing the Arg448Lys polymorphism. As expected, the heterozygous control showed equal proportions of the Lys and Arg forms at 928 and 935 m/z, respectively. However, the index case was phenotypically homozygous for Arg448 (there was no 928 m/z signal and the 935 m/z ion was twice the intensity), suggesting that Lys448 is cis to the Gly264 variant and therefore not expressed. The mutated residue 264 is located in peptide D-15 (DFGRKW) and the predicted mass of the M+1H ion of this peptide is 808 m/z. This ion appeared at similar intensity in both maps, suggesting that variant chains containing the DFGGKW peptide were not present. BβArg264 is located in the BβD domain, the importance this domain in viable protein expression has previously been highlighted by other cases of hypofibrinogenaemia and afibrinogenaemia. For example, the heterozygous Bβ Arg255His mutation in fibrinogen Merivale is in close proximity to Bβ Arg264 and was associated with an average plasma fibrin concentration of