| Objective:Hereditary coagulation factor deficiency is one of the most common reasons of hemorrhagic diseases,and its clinical manifestations are mainly bleeding tendency with different degrees.At present,there is no mature cure therapy available for clinical except for the replacement therapy by infusion of corresponding coagulation factors.The main pathogenesis of hereditary coagulation factor deficiency is the genetic variation in the corresponding coagulation factor,leading to defects in the quantitative or function of coagulation factors.Common hereditary factor deficiencies are hemophilia A,hemophilia B and von Willebrand disease,while rare bleeding disorders include the inherited deficiencies of fibrinogen,factor II,factor V,factor VII,factor X,factor XI and factor XIII.In the diagnosis of hereditary coagulation factor deficiency,the routine tests can clearly diagnose the patients with severe bleeding and a family history.However,it is difficult to use routine tests to confirm the diagnosis of the patients with a moderate to mild phenotype or no family history.Based on this,molecular diagnosis has certain advantages in distinguishing phenotypes and providing genetic diagnosis,and can provide a theoretical support for clinical bleeding management.This study is aim to perform molecular diagnosis of factor X（Factor X,FX）deficiency,hemophilia A（HA）,and hemophilia B（HB）,and it is planned to confirm the disease phenotype and define the genetic information.Moreover,this research explored the pathogenic mechanism of novel genetic variation,and supplement the variants spectrum of coagulation factor deficiency and disease spectrum.Methods:1.The phenotype diagnosis of FX deficiency,HB and HA Coagulation factor activity and antigen were detected by a one-stage method and ELISA,respectively.FVIII/FIX inhibitor was tested using the Bethesda method.2.The molecular diagnosis of FX deficiency,HB and HA The molecular diagnosis of FX deficiency and HB were performed by PCR and Sanger sequencing.The sequencing results were compared with public databases to confirm the sequence variation of FX and FIX gene.The molecular diagnosis of HA started with the inversions in intron detection of HA.Long distance PCR（LD-PCR）and double-tube multiplex PCR were used to detect inversions in intron 22（IVS22）and intron 1（IVS1）,respectively.The patients whom resulted negative for IVS22 and IVS1 were directly analyzed by NGS.The sequencing results were further confirmed by gene annotation analysis and Sanger sequencing to confirm the variants site.3.The pathogenic mechanism of novel variants The effect of variant on protein function was analyzed,by constructing overexpressed wild type FX gene vector and mutant FX gene vector that was transfected into 293 T cells.After that,the impact of variation on protein structure and function was investigated by using bioinformatics analysis including homology modeling and molecular dynamics simulation.Through SIFT and Poly Phen-2 to calculate sequence homology and potential pathogenicity of missense mutations,the pathogenic mechanism on novel variants of HA and HB was studied.The splice variation is predicted by human splice finder version 3.1（HSF,http://www.umd.be/HSF）to analyzed the effect of FIX variants.The SIFT and Poly Phen-2 were used to analyze the damage of missense mutation to the structure and function of FIX and FVIII.The Homology modeling of FIX protein structure was built using i-Tasser software,and drawn using the Py MOL program.The Swiss-model software and the tool of Swiss-Pdb Viewer were used to build the he homology modeling of the mutated FVII protein,and to display and map the modeled molecules.Results:1.Genetic analysis revealed that the proband of FX deficiency and his affected sister were homozygous for c.1085G>A mutation,corresponding to a Ser362 Asn substitution.In vitro expression experiments showed that the FX Ser362 Asn mutation led to a significant reduction in activity levels in the culture medium.This Ser to Asn substitution may change the shape of the active site.Moreover,simulations of molecular dynamics indicated that the binding energy of the FX Ser362 Asn to the substrate is higher than that of wild type and the side-chain conformation of the catalytic residue His276（His42）is changed.2.A total of 23 variants were detected in 32 HB patients.Among them,18 variants were included in the F9 gene variant database and 5 variants were novel.Novel variants are c.277 + 5 G> T,c.723 + 1G> A,c.839-6c.839-5 ins,c.553557 del CAAAC（p.Gln185 Phefs * 1）and c.1215T> G（p.Asp405Glu）.The splice site variants c.277 + 5 G> T and c.723 + 1G> A mainly disrupt the recognition of normal splice sites;the of large insertion,c.839-6c.839-5 ins located near the splice site,destroy the correct splicing sites and form multiple abnormal splicing points;the small deletion,p.Gln185 Phefs * 1,is mainly due to the frameshift effect to generate truncated non-functional F9 protein;missense mutation p.Asp405 Glu is located in the F9 catalytic region,and it has a certain influence on the structure and function of FIX protein.3.A total of 53 HA patients were included and 4 samples were not found gene variants of F8 or other coagulation factor genes.The F8 variants include intro 22 inversion（31 samples）,intron 1 inversion（3 samples）,9 missense,2 nonsense and 4 cases of frameshift（2 cases of deletion,1 case of insertion,1 case of single base duplication）.Except for the single base G duplication variant（p.Ile1213Asnfs*28）which was the hot spot mutation reported by the research group in the early stage,the rest of the variants were found for the first time in the region.The missense variants p.Cys172 Ser,p.Tyr404 Ser,p.Asp1903 Gly,p.Ser2284 Asn,the deletion p.Leu2249fs*9 and the insertion p.Pro2319fs*97 are the novel variants.In the F8 variants,due to Cys172 being in a highly conserved disulfide bond structurein the A1 domain,the missense mutation p.Cys172 Ser results in severe HA.The p.Asp1903 Gly affects the correct conformation of the A3 domain of FVIII protein to a certain extent,which further influences the binding to VWF,leading to FVIII protein dysfunction and the decreased activity.The effect of the p.Ser2284 Asn on FVIII protein is that the hydrogen bonds around Asn2284 increase,and the stability of the protein structure decreases.The protein structure of the p.Tyr404 Ser is shown that the conformation of amino acid residues has changed a lot with Ser substitution Tyr,with the decreased hydrogen bond and the enhanced hydrophobicity. The p.Leu2249 fs * 9 is due to the presence of a base G deletion in exon 25,resulting in amino acid frameshifting and the loss of the entire functional domain encoded by exon 26.The small insertion variant p.Pro2319 fs * 97 is caused by the frame-shifting effect,so that the translation process of the FVIII protein is not terminated normally,and the structure of the FVIII protein is greatly changed.Conclusion:1.Our findings suggest that the Ser362 Asn substitution is a pathogenic mutation that causes inherited FX deficiency.This impairs the conformational switch of the protein from zymogen to proteinase,thus causing the functional defect of FX protein.2.The spectrum of F9 variants in the regions confirmed the remarkable heterogeneity of F9 gene variants.Direct sequencing is still a powerful and cost-effective technique for detecting variants in the F9.Missense mutation is the most common type of mutation,which mainly occurs in the catalytic region of F9.3.The study of the F8 genetic variants in this region has found that there is a large heterogeneity.The application of NGS molecular diagnosis enriches the variant spectrum of HA,which is of great significance for the individualized genetic counseling and clinical diagnosis and treatment.NGS and a variety of bioinformatics prediction methods can further analyze the impact of genetic variation on protein structure or function,and lay the foundation for revealing the molecular pathogenic mechanism of novel variants. |
