Study On The Molecular Pathogenesis Of Skewed X Chromosome Inactivation Leads To Female Hemophilia And Diagnosis On The Phenotype And Genotype Of 6 Inherited Factor X Deficiency

Objective: Skewed X chromosome inactivation is one of the main pathogenesis of female hemophilia.However the mechanism of Skewed X chromosome inactivation is unclear.Here,we mainly study the molecular pathogenesis of female hemophilia caused by Skewed X chromosome inactivation.Methods: Coagulation assays were performed to establish the phenotypes of probands.A serial of genetic tests were perfomed to analyze F8,F9 and XIST,including including directly sequencing the PCR amplification products of F8,F9 and XIST,detection of copy number variations(CNVs)and detection of intron 1and 22 inversion,F8 or F9 gene linkage analysis,analysis of X inactivation percentage(XIP)using both a classical human androgen receptor(HUMARA)assay and RP2 assay,karyotype and High-density single nucleotide polymorphism(SNP)genotyping array analysis were utilized.X-chromosome gene linkage analysis to locate pathogens related to skewed X chromosome inactivation.Results: Here,we reported 6 HA patients and 3 HB patients.Genetic analysis showed that the proband HA1 presented with mutation c.1063C>T,p.Arg355 *,and the proband HA2 had a mutation c.6241T> G,p.Trp2081 Gly The proband HA3 had a mutation c.5998 + 1,G> C,the proband HA4 showed a d a large heterozygous deletion of exon 2-exon 6 in F8 gene.The proband HA5 F8 showed a large duplication of exon 2-exon 26 in F8 gene.The proband HA6 was the carrier of intron 22 inversion.In 3 patients with hemophilia B,genetic diagnosis showed that the proband HB1 presented with a mutation c.508T> C,p.Cys170 Arg,proband HB2 had a mutation c.1226G> A,p.Gly409 Glu,the proband HB3 presented with mutation c.255 + 5G> A.Analysis of X inactivation percentage(XIP)showed that 7 of 9 patients had skewed X chromosome inactivation.A negative linear relationship between FVIII or FIX activity and percentage-inactivated mutanted X-chromosome was found in HA or HB heterozygous females.XIST gene analysis showed that 3 female patients had XIST mutations,but these three mutations were not related to the skewed X chromosome inactivation.A copy number variant located on Xq28 were revealed by the array analysis.A 2904 kb deletion(arr[hg19] Xq28(152,328,908-155,233,098)x1)covering more than 60 OMIM genes in proband HB3 were categorized as pathogenic according to widely accepted guidelines.HA1 family’s X chromosome linkage analysis showed that a locus for familial skewed X chromosome inactivation maps to chromosome Xp21.2Xp11.4.Conclusion: A negative linear relationship between FVIII or FIX activity and percentageinactivated mutanted X-chromosome was found in HA or HB heterozygous females.A2904 kb deletion(arr[hg19] Xq28(152,328,908-155,233,098)x1)covering more than 60 OMIM genes in proband HB3 were categorized as pathogenic.It is speculated that the Xq28 deletion will cause cells survival disadvantages,which result in skewed X chromosome inactivation.HA1 family’s X chromosome linkage analysis showed that a locus for familial skewed X chromosome inactivation maps to chromosome Xp21.2Xp11.4,where genetic elements which control X chromosome inactivation exsit or a gene mutation will cause cell selection.Objective: To investigate the molecular pathogenesis of six pedigrees with inherited factor X(FX)deficiency and to assess the bleeding risks in patients with inherited FX deficiency using thrombin generation test(TGT).Methods: Six pedigrees’ clinical data was collected.The FX coagulation activity(FX:C)and antigen(FX:Ag)were tested by clotting test and ELISA method.The FX in the plasma was measured with western blotting.The factor X gene(F10)defects were analyzed by direct sequencing.Thrombin generation(TG)in six patients and some family members was measured using a standard procedure with commercial reagents.Results: Ten F10 mutations were identified in six probands with FX deficiency,five of which(Cys246Ser,Tyr319 Cys,Leu252Phe,Arg313 Gln and IVS5-2A>G)were novel and another five(Phe71Ser,Val338 Met,Gly406Ser,Gly365 Ser and Cys151Arg)have been previously reported.TGT showed that there is correlation between parameters of endogenous thrombin potential(ETP)and peak height(Peak)and the clinical expressions of the FX-deficient patients.Conclusions: Six probands were diagnosed as inherited FX deficiency.Five of them were resulted from compound heterozygous F10 mutations,while the remaining one was caused by one heterozygous F10 mutation.TGT parameters of endogenous thrombin potential(ETP)and peak height(Peak)can be used to assess the bleeding risks in patients with FX deficiency and TGT may serve as a useful laboratory tool to prevent and treat the disease.