title: Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bbeta-chain gene causing activation of cryptic splice sites.
abstract: 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 Aalpha, 6 in the gamma, and only 2 in the Bbeta 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 Bbeta-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bbeta-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 Bbeta-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 Bbeta chain for fibrinogen assembly and secretion.
authors.affiliation: Department of Biology and Genetics for Medical Sciences, University of Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Spena
authors.forename: Stefano
authors.initials: S
authors.lastname: Duga
authors.forename: Rosanna
authors.initials: R
authors.lastname: Asselta
authors.forename: Massimo
authors.initials: M
authors.lastname: Malcovati
authors.forename: Flora
authors.initials: F
authors.lastname: Peyvandi
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Tenchini
mesh_terms: D000293:Adolescent; D000347:Afibrinogenemia / Q000235:genetics*; D001483:Base Sequence; D001780:Blood Coagulation Tests; D002675:Child, Preschool; D003001:Cloning, Molecular; D003241:Consanguinity; D004252:DNA Mutational Analysis; D005340:Fibrinogen / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D006367:HeLa Cells; D006474:Hemorrhagic Disorders / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D007492:Iran; D007558:Italy; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010375:Pedigree; D011487:Protein Conformation; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: C115499:BBeta fibrinogen; D022821:RNA Splice Sites; D005340:Fibrinogen