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 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.
Asselta
R
R
Department of Biology and Genetics for Medical Sciences, University of Milan; Institute of Veterinary Physiology and Biochemistry, University of Milan, Italy.
Duga
S
S
Simonic
T
T
Malcovati
M
M
Santagostino
E
E
Giangrande
P L
PL
Mannucci
P M
PM
Tenchini
M L
ML
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
D012333:RNA, Messenger; D005340:Fibrinogen