| Objective:1.To make genetic diagnosis for a family with hemophilia B(HB).2.To study the molecular pathogenesis of the novel missense variant c.1181T>C(p.Met394Thr).Methods:1.Blood coagulation tests: activated partial thromboplastin time(APTT)and prothrombin time(PT)were measured by coagulation method.The activity of factor Ⅸ(FⅨ:C)was measured by one-stage method.The content of factor Ⅸ antigen(FⅨ:Ag)was measured by enzyme-linked immunosorbent assay.2.Gene variant analysis: the coding region and its flanking sequences of coagulation factor Ⅸ gene(F9)were amplified by PCR,and then sanger sequencing was used to detect the variation of F9.3.In vitro experiments: To explore the molecular pathogenesis of the new mutation,we performed in vitro experiments.Plasmids containing wild-type or mutant coagulation factor Ⅸ(FⅨ)c DNA were transiently transfected into HEK293 T cells,followed by detection of FⅨ gene(F9)m RNA expression levels using quantitative real-time fluorescence PCR(q RT-PCR);The FⅨ antigen(FⅨ:Ag)content was detected by enzyme-linked immunosorbent assay(ELISA);the FⅨ protein content was detected using Western Blotting.4.Bioinformatics analysis: polyphen2,SIFT and Mutation Taster were used to predict the pathogenicity of novel variant;The homologous sequences from different species were aligned by Unipro UGENE software to evaluate the conservation of amino acid residues at different sites of FⅨ protein;The wild-type FⅨ protein was modeled using the Swiss-model online tool and visualized using Pymol software.Results:1.Coagulation test showed that the proband and his brother had prolonged APTT(43s and 37.4s)and normal PT(12.3s and 11.2s).FⅨ:C was 1.9% and 2.9% in the proband and his brother,respectively.The APTT of the proband’s grandmother was slightly prolonged,FⅨ: C was 31.12%,and the APTT of the proband’s mother was normal,FⅨ: C was 50.2%.The results of coagulation testing of the proband’s father were normal.2.Results of gene analysis: we identified a novel missense variant(c.1181T>C,p.Met394Thr)in the HB family,the proband’s mother and grandmother were carriers of the variant.In addition,grandmother F9 had a point variant(c.88+75A>G)in intron 1that may affect the function of the FⅨ protein.3.The results of in vitro experiments showed that when the levels of wild type FⅨ:C and FⅨ: Ag were set to 100%,the FⅨ: C in the culture medium of the mutant(FⅨ-Met394Thr)cells was 9.3%±4.4%.The results of ELISA showed that the levels of FⅨ: Ag in the culture medium and lysate of the mutant cells were 87%±18% and98%±3%,respectively.Western Blotting showed that there was no significant difference in the protein content in or outside the cells compared with the wild type.4.The results of bioinformatics analysis: using polyphen2,SIFT and Mutation Taster,the p.Met394 Thr variant was predicted to be pathogenic;Conservation analysis showed that methionine at position 394 of FⅨ protein was highly conserved in multiple species;Model analysis showed that the structure of the mutant amino acid residues and their surrounding hydrogen bonds were significantly changed.In addition,the p.Met394Thr variant was spatially close to the disulfide bond formed by Cys382 and Cys396,and this variant may affect the disulfide bond formation.Conclusion:1.This study reported a novel variant in F9,which enriches the F9 mutation database.2.p.Met394 Thr variant did not affect F9 transcription,FⅨ protein synthesis or secretion.This variant may cause HB by destroying the spatial conformation of FⅨ protein.3.This study may provide new strategies for future precision treatment of HB... |
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