Novel fibrinogen mutations (Aα17Gly→Cys and Aα381Ser→Phe) occurring with a 312Thr→Ala polymorphism: allelic phase assigned by direct mass measurement

Abstract

The aim of the study was to determine the molecular cause of dysfibrinogenaemia in a woman with a prolonged thrombin time. Functional fibrinogen abnormalities can be benign or may lead to bleeding or thrombotic conditions. In complex cases, phenotypes may be acquired or involve interplay between several coinherited mutations. The authors developed a new whole-protein time-of-flight mass spectrometry (TOF MS) approach to direct targeted DNA sequencing of the fibrinogen genes and determine the phase of multiple substitutions in a single individual. TOF MS analysis of the individual's fibrinogen indicated normal B[beta], [gamma], and alternately transcribed [gamma]' chain isoforms, but aberrant A[alpha] chain masses. Subsequent fibrinogen A[alpha] gene (FGA) sequencing indicated the presence of three different mutations. Two of the substitutions, A[alpha]17Gly->Cys (at the thrombin cleavage site) and A[alpha]381Ser->Phe (in the [alpha]C connector) were novel and the third, A[alpha]312Thr->Ala, was a known benign polymorphism. Accurate mass measurements of isolated control A[alpha] chains showed the predicted A[alpha] polypeptide at 66 132 Da and additional phosphorylated species at + 80 and + 160 Da. Patient's A[alpha] chains on the other hand had masses of 66 103 and 66 241 Da indicating that she had one 312Ala allele (-30 Da) and one 312Thr allele which carried both the 17Gly->Cys (+ 46 Da) and 381Ser->Phe (+ 60) Da mutations. Cotransmission of these new mutations was confirmed by A[alpha] chain TOF MS of plasma fibrinogen and targeted FGA nucleotide sequencing for 10 additional family members.