Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bbeta-chain gene causing activation of cryptic splice sites

Blood. 2002 Dec 15;100(13):4478-84. doi: 10.1182/blood-2002-06-1647. Epub 2002 Aug 1.

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.

Publication types

  • Case Reports
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Afibrinogenemia / genetics*
  • Base Sequence
  • Blood Coagulation Tests
  • Child, Preschool
  • Cloning, Molecular
  • Consanguinity
  • DNA Mutational Analysis
  • Fibrinogen / genetics*
  • Genetic Predisposition to Disease
  • HeLa Cells
  • Hemorrhagic Disorders / genetics
  • Humans
  • Introns / genetics
  • Iran
  • Italy
  • Male
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Pedigree
  • Protein Conformation
  • RNA Splice Sites / genetics*
  • RNA Splicing / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription, Genetic
  • Transfection

Substances

  • BBeta fibrinogen
  • RNA Splice Sites
  • Fibrinogen