Study On The Molecular Pathogenesis Of Hemophilia A Caused By Two Splice Site Mutations Of F8 And Hemophilia B Caused By Two Large Duplications Of F9

Hemophilia A/B is an X-linked inherited bleeding disorder caused by quantitative and qualitative defects of factor ? or F? associated with F8/F9 mutations.Objective To study the molecular pathogenesis of two novel splice site mutations of F8.Methods Coagulation assays were performed to establish the phenotypes of probands A.A serial of genetic tests were performed to analyze F8,including directly sequencing the PCR amplification products of 26 exons and flanking regions of F8,detection of copy number variations?CNVs?and detection of intron 1 and 22 inversion.Nested PCR was applied to detect F8 ectopic transcripts in leukocyte to analyze the molecular pathogenesis of the splice site mutations.Multiplex fluorescent PCR was used to detect six STR locus for gene linkage analysis.Mosaic analysis was performed in peripheral blood,oral mucosal cells and hair follicle cells by SNaPshot SNP technique to analyze the origin of mutation.Results Levels of F?:C were 0.9% and 5.1% in probands 1 and 2 respectively.Proband 1 presented with a splice site mutation in intron 9?c.1444-2dupA?.mRNA analysis showed a smaller band,resulting from exons 10 and 11 skipping.Proband 2 had a T>G substitution in intron 19?c.5999-29T>G?.mRNA analysis showed a smaller band and a weak normal size band.The smaller band presented exon 19 skipping.In pedigree 1,the mutation was inherited from proband's grandfather who was with normal phenotype and genotype.Mosaic analysis showed no mosaic existing in his peripheral blood,oral mucosal cells and hair follicle cells.Conclusions Mutations of c.1444-2dupA and c.5999-29T>G caused severe and mild hemophilia A respectively.c.1444-2dupA in pedigree 1 was a de novo mutation which may arose from sperm of proband's grandfather at meiosis.Objective To study the molecular pathogenesis,recombination mechanisms and origin of two novel duplications of F9.Methods Plasma F? protein was analyzed,like F? activity,F? antigen and western blotting,to figure out the effect of large duplications.Breakpoint detection was carried out by long-range PCR,primer walking methods combined with genome walking strategy respectively.In the meantime,multiple fluorescent PCR was used to detect the mosaic rate of large duplication and analyzed the origin of the mutations in pedigree B.In addition,bioinformatics was performed to analyze recombination mechanism.Results We found that F? activity and antigen were about 8% in proband A.There was a small amount of normal F? protein in plasma.Duplication of exons 1-6 in F9?g.139,518,026₁39,551,945dup?was detected in proband A with same direction in tandem.F? activity and antigen were <1% and 3.1% respectively in proband B.A small amount of large F? was found in plasma of proband B.Duplication of exons 4-6 in F9?g.139,537,677₁39,551,836dup?was detected in proband B with same direction in tandem.In vitro mRNA analysis of minigene showed that three transcripts could be expressed in proband B,of which one transcript?about 3%?did not lead to the reading frame shift.Cellular proportions of mosaic were 6.66% in peripheral blood,0% in hair follicle cells,14.10% in oral mucosal cells and 13.22% in uroepithelial cells of the grandmother.Bioinformatics analysis showed that there were some repeat elements which related to the duplication formation near the breakpoints of the two large duplications,while no homologous sequences existed.Conclusions We speculated that two large duplications were the causative reasons of the two families respectively.Large duplications may be formed by the NHEJ and MMEJ recombination mechanisms respectively.The mutation arose in embryonic development of proband B' grandmother,which affected some of its somatic and germ cells.The possible pathogenic mechanisms of two large duplications are as follows: Large duplication might affect function of promotors by promotor competition;and large duplication might affect the normal recognition of spliceosomes.