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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-06-1647.
Submitted June 4, 2002
Accepted July 22, 2002
Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bß-chain gene causing activation of cryptic splice sites
Silvia Spena, Stefano Duga, Rosanna Asselta, Massimo Malcovati, Flora Peyvandi, and Maria Luisa Tenchini*
Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, Department of Internal Medicine, University of Milan and IRCCS Maggiore Hospital, Milan, Italy
* Corresponding author; email: marialuisa.tenchini{at}unimi.it.
Congenital afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in afibrinogenemia, 17 in the A , 6 in the  , and only 2 in the Bß fibrinogen-chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bß-chain gene (IVS6+13C>T and IVS7+1G>T), representing the first Bß-chain gene splicing mutations described in afibrinogenemia. The IVS6+13C>T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7+1G>T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bß-chain minigene constructs, either wild type (wt) or mutant, were transfected in HeLa cells. Assessed by semi-quantitative analysis of RT-PCR products, the IVS7+1G>T mutation resulted in multiple aberrant splicings, while the IVS6+13C>T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bß-chain for fibrinogen assembly and secretion.
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