Molecular pathogenesis of the patients with congenital disorder of thrombosis and hemostasis

I.Analysis of Bernard-Soulier syndrome：Phe I D155锡D1(TTT)to Ser(TCT)replacement in the leucine rich motif(LRM)of the GPIX polypeptide does cause BSS phenotype.We have certified this by means of in vitro transfection studies with plasmid for mutant GPIX and other plasmids for GPIb/IX complex。Mutant GPIX could not increase the surface expression of GPIb-αnor surface expression of GPIX itself.II.Analysis of coagulation factor X deficiency：We have identified the molecular defect underlying congenital factor X(FX)deficiency.A novel3-base-pair deletion was observed within intron D of the FX gene.This mutation was not detected in 53 unrelated Japanese(106 Alleles)by an allele-specific restriction analysis，indicating a likely cause of the FX deficiency。The deletion resides within a polypyrimidine tract of the acceptor splicing site where U2snRNP binds to form spliceosomes。This defect could alter the formation of splicesomes，then result in incorrect splicing and decre…More ased FX production.III.Analysis of coagulation factor XI deficiency：We have identified a novel single-base point mutation，a GT-&gt；AT transition of the factor XI(FXI)gene at the donor splicing site of its intron J.A reverse transcription-polymerase chain reaction with ectopic RNA revealed that propositus cDNA showed20-base truncation of the3‘-end of the FXI exon10，resulting in the frame-shift and generation of the stowt.A phenotypic consequence of this mutation was then investigated through mammalian cell expression system。Recombinant plasmids harboring wild type FXI gene did direct the production of the FXI antigen into the medium，but the plasmids lacking20bases of the FXI exon10 failed to produce the antigen while the mRNA expression thereof was unchanged。The mutation described here is novel，and ectopic RNA from peripheral mononuclear cells was first proved to be useful for analyzing the resultant FXI mRNA sequence.Less：Less

I. Analysis of Bernard-Soulier syndrome : PheィイD155ィエD1 (TTT) to Ser (TCT) replacement in the leucine rich motif (LRM) of the GPIX polypeptide does cause BSS phenotype. We have certified this by means of in vitro transfection studies with plasmid for mutant GPIX and other plasmids for GPIb/IX complex. Mutant GPIX could not increase the surface expression of GPIb-α nor surface expression of GPIX itself.II. Analysis of coagulation factor X deficiency : We have identified the molecular defect underlying congenital factor X (FX) deficiency. A novel 3-base-pair deletion was observed within intron D of the FX gene. This mutation was not detected in 53 unrelated Japanese (106 alleles) by an allele-specific restriction analysis, indicating a likely cause of the FX deficiency. The deletion resides within a polypyrimidine tract of the acceptor splicing site where U2 snRNP binds to form spliceosomes. This defect could alter the formation of splicesomes, then result in incorrect splicing and decre … More ased FX production.III. Analysis of coagulation factor XI deficiency : We have identified a novel single-base point mutation, a GT->AT transition of the factor XI (FXI) gene at the donor splicing site of its intron J.A reverse transcription-polymerase chain reaction with ectopic RNA revealed that propositus cDNA showed 20-base truncation of the 3'-end of the FXI exon 10, resulting in the frame-shift and generation of premature stop codon at 56-base downstream of the correct exon 10/11 junction. A phenotypic consequence of this mutation was then investigated through mammalian cell expression system. Recombinant plasmids harboring wild type FXI gene did direct the production of the FXI antigen into the medium, but the plasmids lacking 20 bases of the FXI exon 10 failed to produce the antigen while the mRNA expression thereof was unchanged. The mutation described here is novel, and ectopic RNA from peripheral mononuclear cells was first proved to be useful for analyzing the resultant FXI mRNA sequence. Less