Studies On The FIX Gene Mutation And Molecular Pathogenesis Of Hemophilia B

Objective：To study the phenotype and genotype in three inherited coagulation factor Ⅸ（FⅨ）deficiency families that were given informed consent by clinical examination andpedigree analysis, and identify causative gene defects in all known patients with hemophiliaB（HB）in Chinese Han people. To strive to reveal its pathogenesis at the molecular level andget a better understanding of HB.Methods: Activated partial thromboplastin time （APTT）, prothrombin time （PT）, thrombintime （TT）, fibrinogen （Fg） and coagulation factors F Ⅸ activity （F Ⅸ: C） were tested tomake phenotypic diagnosis. Genomic DNA was extracted from each propositus and thefamily members’ peripheral blood leucocytes. Using primer Premier5software, nine pairsprimers were designed to cover all eight exons areas and the flanking sequences of FⅨGene respectively, according to the FⅨ Gene reference sequences （the GenBank entryNC₀00023.10）. For the propositi and suspicious carriers, all regions of F Ⅸgene, includingall exons and the flanking sequences, were amplified by Polymerase Chain Reaction （PCR）.The products of PCR were sequenced by the dideoxy chain termination using ABI3730XLsequencer. Then the sequencing results were analyzed with software Chromas2andBLAST, searching for mutations in these regions of the gene. Large deletion of the FⅨgene was detected with long distance-PCR （LD-PCR） and agarose gel electrophoresis.Results: By PCR and direct gene sequencing, two missense mutations and a partial genedeletion were identified in F Ⅸgene of the propositi when compared with the reference sequence. We discovered a homozygous missense mutation g.25377C> T, with amino acidchanging Cys into Arg in the exon6of a three-year old girl’s FⅨ gene,which is a relativelyrare case in the world. In thepedigree1,a missense mutation g.15437G> A with amino acidchanging Gly into Glu was identified.An11214bp deletion including exon4,exon5andintron4in a severe hemophilia B patient of the pedigree2was found, which was firstreported in the international. And we localized the breakpoint of the newly discovered largedeletion by segmented PCR and sequencing.Conclusions: The molecular basis for the pathogenesis of hemophilia B is gene mutationsof F Ⅸ.The genotype-phenotype correlations between the type of mutation and the severityof factor deficiency are not entirely consistent. Exon sequencing is one of the straightest,most reliable and most economical methods for the diagnosis of patients and carriers ofHB.