TY - JOUR
T1 - Congenital afibrinogenemia
T2 - First identification of splicing mutations in the fibrinogen Bβ-chain gene causing activation of cryptic splice sites
AU - Spena, Silvia
AU - Duga, Stefano
AU - Asselta, Rosanna
AU - Malcovati, Massimo
AU - Peyvandi, Flora
AU - Tenchini, Maria Luisa
PY - 2002/12/15
Y1 - 2002/12/15
N2 - 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 Aα, 6 in the γ, and only 2 in the Bβ 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 Bβ-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bβ-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 Bβ-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 Bβ chain for fibrinogen assembly and secretion.
AB - 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 Aα, 6 in the γ, and only 2 in the Bβ 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 Bβ-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bβ-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 Bβ-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 Bβ chain for fibrinogen assembly and secretion.
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U2 - 10.1182/blood-2002-06-1647
DO - 10.1182/blood-2002-06-1647
M3 - Article
C2 - 12393540
AN - SCOPUS:0037114754
VL - 100
SP - 4478
EP - 4484
JO - Blood
JF - Blood
SN - 0006-4971
IS - 13
ER -