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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-06-1647.
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Blood, 15 December 2002, Vol. 100, No. 13, pp. 4478-4484
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Congenital afibrinogenemia: first identification of splicing
mutations in the fibrinogen B -chain gene causing activation of
cryptic splice sites
Silvia Spena,
Stefano Duga,
Rosanna Asselta,
Massimo Malcovati,
Flora Peyvandi, and
Maria Luisa Tenchini
From the Department of Biology and Genetics for Medical
Sciences, University of Milan; the Angelo Bianchi Bonomi Hemophilia and
Thrombosis Center and Fondazione Luigi Villa, Department of Internal
Medicine, University of Milan; and the Istituto di Ricovero e Cura a
Carattere Scientifico (IRCCS) Maggiore Hospital, Milan,
Italy.
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 B
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 B-chain gene (IVS6 + 13C > T and
IVS7 + 1G > T), representing the first B-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
B-chain minigene constructs, either wild-type or mutant, were
transfected in HeLa cells. Assessed by semiquantitative analysis of
reverse transcriptase-polymerase chain reaction 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 B chain for
fibrinogen assembly and secretion.
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