title: Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation.
abstract: Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of plasma fibrinogen and by a bleeding tendency ranging from mild to moderately severe. Beside a deletion of the almost entire Aalpha-chain gene, only 2 missense mutations in the C-terminal domain of the Bbeta-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable plasma levels of functional and immunoreactive fibrinogen. Sequencing of the fibrinogen genes revealed a homozygous G-->A transition at position +5 of intron 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 role 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 transfection of the mutated plasmid in HeLa cells, followed by RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowed us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicing occurred in HeLa cells transfected with the wild-type plasmid. This is the first report of a mutation in the fibrinogen gamma-chain gene causing afibrinogenemia and indicates that, in addition to the Aalpha and Bbeta-chain genes, the gamma-chain gene must also be considered in mutation screening for afibrinogenemia.
authors.affiliation: Department of Biology and Genetics for Medical Sciences, University of Milan; Institute of Veterinary Physiology and Biochemistry, University of Milan, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Asselta
authors.forename: S
authors.initials: S
authors.lastname: Duga
authors.forename: T
authors.initials: T
authors.lastname: Simonic
authors.forename: M
authors.initials: M
authors.lastname: Malcovati
authors.forename: E
authors.initials: E
authors.lastname: Santagostino
authors.forename: P L
authors.initials: PL
authors.lastname: Giangrande
authors.forename: P M
authors.initials: PM
authors.lastname: Mannucci
authors.forename: M L
authors.initials: ML
authors.lastname: Tenchini
mesh_terms: D000347:Afibrinogenemia / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002675:Child, Preschool; D003241:Consanguinity; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics*; D006367:HeLa Cells; D006470:Hemorrhage / Q000235:genetics; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D010154:Pakistan; D010375:Pedigree; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D005340:Fibrinogen