Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation

Congenital afibrinogenemia is a rare autosomal recessive disorder character ized by the complete absence of plasma fibrinogen and by a bleeding tendenc y ranging from mild to moderately severe. Beside a deletion of the almost e ntire A alpha-chain gene, only 2 missense mutations in the C-terminal domai n of the B beta-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable pla sma levels of functional and immunoreactive fibrinogen, Sequencing of the f ibrinogen genes revealed a homozygous G-->A transition at position +5 of in tron 1 of the gamma-chain gene. The predicted mutant fibrinogen gamma-chain would contain the signal peptide, followed by a short stretch of aberrant amino acids, preceding a premature stop codon. To demonstrate the causal ro le of the identified mutation, we prepared expression vectors containing a region of the fibrinogen gamma-chain gene spanning from exon 1 to intron 4 and carrying either a G or an A at position +5 of intron 1, Transient trans fection of the mutated plasmid in HeLa cells, followed by RNA extraction an d reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowe d us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicin g occurred in HeLa cells transfected with the wild-type plasmid. This is th e first report of a mutation in the fibrinogen gamma-chain gene causing afi brinogenemia and indicates that, in addition to the A alpha and B beta-chai n genes, the gamma-chain gene must also be considered in mutation screening for afibrinogenemia. (Blood. 2000;96: 2496-2500) (C) 2000 by The American Society of Hematology.