Four New Genetic Mutations Lead To The Molecular Pathogenesis Of Research And Read Through The Therapy Of Hemophilia B Nonsense Mutation Hemophilia Treatment Due To The Initial Research

The First Part The Preliminary Studies on the Molecular Pathogenesis of Hemophilia B caused by four new mutationsObjective:1. In order to screen the mutations of the FIX gene in hemophilia B patients and obtain a more comprehensive analysis of the molecular pathology of hemophilia B, the all regions of FIX gene, including all exons and the flanking sequences, were amplified by polymerase chain reaction (PCR).2. This study was designed to construct pIRES2-ZsGreenl/FⅨwt expression vector, thus to provide experiment basement for the follow study on the location, function and molecular pathology of hemophilia B.3. The FⅨ expression levels of the three new mutation, including R116Stop combinded A-38G, C51G and6479insert C, were detected to evaluate their roles in the pathogenesis of hemophila B.4. The FIX expression levels of the new insertional mutagenesis, FIX17784insert103bp, were detected to evaluate its role in the pathogenesis of hemophila B.Method:1. The peripheral blood samples were extracted from11unrelated hemophilia B probands and their pedigrees members. All the8exons and the flanking sequences, were amplified by PCR. The products of PCR were sequenced by the dideoxy chain termination using ABI3730sequencer.2. The sequence results were compared with normal sequences of FIX gene using Chromas for finding the mutations. For the missense mutation, the corresponding region of FIX gene of100health controls were amplified to rule out the polymorphism.3. The total ORF of FIX gene was amplified from pcDNA/FⅨwt plasmid, then the amplified fragment was clonded into the pIRES2-ZsGreenl vector which were enzymed by double restriction with EcoR I and BamH I. The eukaryotic expression vector of pIRES2-ZsGreen1/F IXwt was constructed and confirmed by PCR and sequencing.4. Using short tandem repeat (STR) technique detected the5exon region of the probands and their pedigrees members with the new insertional mutagenesis, FIX17784insert103bp. 5. The expression vectors, carrying the four new mutations found in the FIX gene of the hemophilia B patients, were constructed by site-directed mutagenesis and confirmed by PCR and sequencing.6. Transient expression experiments were performed using HEK-293cells transfected with the expression vectors containing the wild-type or the mutation recombinant cDNA and observed the expression of ZsGreenl protein by confocal laser microscope.7. The relative expression levels of FIX mRNA of the wild-type and mutation recombinant cDNA were detected by real time quantitative PCR(RQ-PCR).8. The FⅨ:Antigen (FⅨ:Ag) of wild-type and different mutations protein were tested by enzyme-linked immunosorbent assay(ELISA).9. The FⅨ:Activity (FⅨ:C) of wild-type and different mutations protein were tested by One-Stage method.10. The expression level of FIX of wild-type and different mutations protein were tested by immunofluorescence microscopy.Result:1. The sequence results of the11unrelated hemophilia patients revealed11types of gene mutations, including8point mutations in the exons regions (R116Stop combinded A-38G, VI81A, C336R, C51G, R-4Q, A-21D, F32S, C361R), I point muatation in the flanking sequences (G20566A (G→A)),1single nucleotide insertional mutagenesis (6479insert C) and1103bp insertional mutagenesis (FIX17784insert103bp).2. There were four novel mutations in the total11mutaions, including R116Stop combinded A-38G, C51G,6479insert C and FIX17784insert103bp. For the missense mutation, the corresponding region of FIX gene of100health controls were amplified to rule out the polymorphism.3. Construct pIRES2-ZsGreen1/FIXwt expression vector successfully, thus to provide experiment basement for the follow study on the location, function and molecular pathology of hemophilia B.4. The results of STR of the probands and their pedigrees members with the new insertional mutagenesis, FIX17784insert103bp, showed that the probands had four fragment (130.180.233.283bp) and the mutation was from maternal. Meanwhile, the probands had more percentages of normal fragment had less hemorrhage sign.5. The expression vectors, carrying the four new mutations found in the FIX gene of the hemophilia B patients, were successfully constructed.6. Compared with the wild-type FIXmRNA, the relative expression levels of FIX mRNA of R116Stop combinded A-38G were significantly decreased (p<0.05). The analysis of FⅨ:Ag and FⅨ:C in culture media and cell lysates showed apparently decreased compared with the wild-type FⅨ protein(p<0.05). Meanwhile,the immunofluorescence microscopy showed the same results (p<0.05)7. Compared with the wild-type FⅨmRNA, the relative expression levels of C51G FⅨ mRNA of were significantly decreased (p<0.05). The analysis of FⅨ:Ag and FⅨ:C in culture media showed apparently decreased compared with the wild-type FⅨ protein(p<0.05), while there was no difference in the cell lysates (p>0.05). Meanwhile,the immunofluorescence microscopy showed no difference (p>0.05)8. Compared with the wild-type FⅨmRNA, the relative expression levels of6479InsertC FⅨ mRNA of were significantly decreased (p<0.05). The analysis of FⅨ:Ag and FⅨ:C in culture media and cell lysates showed apparently decreased compared with the wild-type FⅨ protein(p<0.05). Meanwhile,the immunofluorescence microscopy showed the same results (p<0.05)9. Compared with the wild-type FⅨmRNA, the relative expression levels of FⅨ17784insert103bp FⅨ mRNA of were no significantly decreased (p>0.05). The analysis of FⅨ:Ag and FⅨ:C in culture media and cell lysates showed apparently decreased compared with the wild-type FⅨ protein(p<0.05). Meanwhile,the immunofluorescence microscopy showed the same results (p<0.05)Conclusion:1. The defect of FⅨ gene is the molecular pathogenesis of HB. The mutations of FⅨ located in any region of the FⅨ gene and the most common mutation was point mutation in the exons regions. Gene direct sequencing is one of the straightest and rigorousest method for the diagnosis of HB.2. R116Stop combinded A-38G mutations could result the production of truncated and non-function FⅨ protein. The mutation was from maternal. Meanwhile, the detection of the gene defect should screen the whole8exons and flanking sequences.3. C51G mutation protein was not been correct folded, by any possibility, the mutation protein had secretion defect. While the obstruction secretion and protein degradation were the molecullar pathology of C51G mutation protein possibly. The mutation was spontaneous mutation.4.6479insert C insertional mutagenesis located in the GLA region of2exon could result the production of truncated protein in the36amino acid, which caused the structure integrity of the GLA region destroyed and influenced the connection between FⅨa and FⅧa. The mutation was from maternal. 5. FIX17784insert103bp caused by the transposon mechanism, which caused the insertion of87nucleotides of5S rRNA. The molecular pathology of this insertional mutagenesis may be the frame shifting of the reading frame and production of truncated protein in the136amino acid, or the mis-shear of the insertional sequences. The mutation was from maternal. The Second Part The Primary Study of Suppression of Premature Termination Codons to Treat Hemophilia Caused by Nonsense MutationsObjective:1. This study was designed to construct pIRES2-ZsGreenl/FⅧ wt expression vector, thus to provide experiment basement for the follow study on the location, function and molecular pathology of hemophilia A.2. Selecting the high frequency nonsense mutations of FⅧ and FⅨ, including (FⅧ:W14X, R1696X, K1827X, R2307X and FⅨ:R29X, R116X, W194X, R333X), as the research objects of the primary study of suppression of premature termination codons to treat hemophilia caused by nonsense mutations.3. Treated the8nonsense mutants with different concentration of chemical compound PTC124and gentamycin to provide the experimental data to the the primary study of suppression of premature termination codons to treat hemophilia caused by nonsense mutations.Method:1. The total ORF of FⅧ gene was amplified from pcl/FⅧwt plasmid, then the amplified fragment was clonded into the pIRES2-ZsGreenl vector which were enzymed by double restriction with EcoR I and BamH I. The eukaryotic expression vector of pIRES2-ZsGreenl/FⅧwt was constructed and confirmed by PCR and sequencing.2. The expression vectors, carrying the eight nonsense mutations in the FⅧ and FⅨ gene, were constructed by site-directed mutagenesis and confirmed by PCR and sequencing.3. Using the cell counting kit-8(CCK-8) detected the cell toxicity of different concentration of chemical compound PTC124and gentamycin to the HEK-293cells and determined the exact concentration of PTC124and gentamycin for the suppression of premature termination codons.4. Transient expression experiments were performed using HEK-293cells transfeeted with the expression vectors containing the wild-type or the nonsense mutation recombinant cDNA and observed the expression of ZsGreenl protein by confocal laser microscope. Meanwhile, treated the8nonsense mutants with different concentration of chemical compound PTC124and gentamycin for48h.5. The relative expression levels of FⅧ and FⅨ mRNA of the wild-type and mutation recombinant cDNA were detected by RQ-PCR.6. The FⅧ:Ag and FⅨ:Ag of wild-type and different mutations protein were tested by ELISA. 7. The FⅧ:C and FIX:C of wild-type and different mutations protein were tested by One-Stage method.Result:1. Construct pIRES2-ZsGreenl/FⅧwt expression vector successfully, thus to provide experiment basement for the follow study on the function and molecular pathology of hemophilia A.2. The expression vectors, carrying the eight nonsense mutations of FⅧ and FⅨ, including (F Ⅷ:W14X, R1696X, K1827X, R2307X and FⅨ:R29X, R116X, W194X, R333X),were successfully constructed.3. The value of half maximal inhibitory concentration of a substance (IC50) of PTC124and gentamycin to the HEK-293cells were111.585±9.597μM and4.23±0.32mM respectively. The concentration of PTC124and gentamycin for the suppression of premature termination codons were10μM,20μM,40μM,60μM,80μM and0.5mM,1.0mM,1.5mM,2.0mM,2.5mM.4. Compared with the wild-type FIXmRNA, FⅨ:W194X, R333X didn't influence the FIXmRNA expression level(p>0.05), the FIXmRNA expression level of FIX:R29X, R116X treated with the PTC124and gentamycin were increased with various degree (p<0.05). Compared with the wild-type FIXmRNA, FⅧ:R1696X didn't influence the FⅧmRNA expression level(p>0.05), the FⅧmRNA expression level of FⅧ:W14X, K1827X, R2307X treated with the PTC124and gentamycin were increased with various degree (p<0.05).5. Compared with the untreated nonsense mutant in cell lysates, the analysis of FIX:C of FIX: R29X, W194X, R116X, R333X treated with2.5mM gentamycin and80μM PTC-124were increased with various degree (p<0.05). Compared with the untreated nonsense mutant in cell lysates, the analysis of FⅧ:C of FⅧ:W14X, R1696X, K1827X, R2307X treated with2.5mM gentamycin and80μM PTC-124were increased with various degree (p<0.05).6. Compared with the untreated nonsense mutant in cell lysates, the analysis of FⅨ:Ag of FⅨ: R29X, R116X, R333X treated with2.5mM gentamycin and80μM PTC-124were increased with various degree (p<0.05), while the FⅨ:W194X with no apparent difference (p>0.05) Compared with the untreated nonsense mutant in cell lysates, the analysis of FVIH:Ag of FⅧ: W14X, R1696X, K1827X treated with2.5mM gentamycin and80μM PTC-124were increased with various degree (p<0.05), while the FVIII:R2307X with no apparent difference (p>0.05)Conclusion:1. One of the most important molecular mechanisms in severe hemophilia is nonsense mutation, carrying premature termination codon(PTC),which lead to the production of nonfunctional truncated protein or the degradation of PTC-bearing mRNA. 2. The IC50value of PTC124to the HEK-293cells were smaller than gentamycin, which may be the result of the cell toxicity of dimethyl sulfoxide (DMSO).3. The expression of FⅧ and FIX mRNA and protein of those nonsense mutant were apparently increased after treated with PTC124and gentamycin. The suppression of PTC to treat hemophilia caused by nonsense mutations of PTC124and gentamycin showed dose-dependent in some degree.4. In the study of suppression of PTC to treat hemophilia caused by nonsense mutations, the PTC124and gentamycin possessed the similar efficiency of read-through. The Third Part The Clinical Significance of Detection of Immune Index in the Patients with Primary Immune ThrombocytopenisObjective:1. Through detecting the frequencies of Circulating B Cells Secreting Platelet-Specific Antibody and Platelet-Specific Antibody in patients with primary immune thrombocytopenia (ITP) and non-immune thrombocytopenia, to evaluate their roles in the diagnosis of ITP and their clinical significance.2. Through detecting the percentage of T lymphocyte subsets in the patients with ITP, to investigate their roles in the cell-mediated immunical pathogenesis of ITP.3. To test the changes of concentration of TPO and the percentage of reticulated platelet in patients with ITP, analyze the correlation between their changes and the platelet count and discuss their role in diagnosis and pathogenesis of ITP.Method:1. The frequencies of Circulating B Cells Secreting Platelet-Specific Antibody and Platelet-Specific Antibody in64ITP patients,33non-immune thrombocytopenia patients and31healthy controls were tested by virtue of Enzyme-linked Immunospot Assay (ELISPOT) and modified Monoclonal antibody immunobilization of platelet antigens assay (MAIPA) respectively.2. The percentage of T Lymphocyte Subsets in64ITP patients and31healthy controls were tested by flow cytometry.3. The level of thrombopoietin and the percentage of reticulated platelet in64ITP patients and31healthy controls were tested by virtue of sandwich enzyme-linked immunosorbent assay and flow cytometry respectively, and PLT(platelet count) and megakaryocyte count were also tested.Result:1. Compared with the controls (1.3±0.5/103PBMC) and non-immune thrombocytopenic purpura patients (2.2±2.0/105PBMC), the frequencies of Circulating B Cells Secreting Platelet-Specific Antibody in newly diagnosed and persistent and chronic ITP were significantly increased (7.6±4.6/105PBMC,5.3±3.0/105PBMC)(P<0.05). However there was no apparent difference of the frequencies of Circulating B Cells Secreting Platelet-Specific Antibody between the controls and non-immune thrombocytopenia patients (P>0.05). The frequencies of Circulating B Cells Secreting Platelet-Specific Antibody in newly diagnosed ITP patients were notably increased(P<0.05) compared to the persistent and chronic ITP patients. Compared with the controls and non-immune thrombocytopenia patients, the Platelet-Specific Antibody in newly diagnosed and persistent and chronic ITP patients were significantly increased (P<0.05). However there were no apparent difference of the Platelet-Specific Antibody between the controls and non-immune thrombocytopenia patients and between the newly diagnosed and persistent and chronic ITP patients (P>0.05). ELISPOT had a sensitivity of68.75%, a specificity of90.91%for the diagnosis of ITP, the sensitivity was higher than that of modified MAIPA's (43.10%)(χ2=6.25, P<0.05).The ROC curve showed the discriminative validity of cytometric bead array was0.885. For the diagnosis of ITP, the sensitivity of ELISPOT was higher than that of modified MAIPA's.2. In T lymphocyte subsets, the percentage of CD3+T Lymphocyte and CD4+T Lymphocyte and the ratio of CD4+/CD8+in patients with ITP (60.88±14.59%,28.41±10.55%,1.18±0.59%respectively) were notably decreased than those healthy controls (of which those of control group were69.89±6.43%,35.38±5.05%,1.64±0.29%respectively, P<0.05), but the percentage of CD8+T Lymphocyte was significantly elevated (which were27.09±9.86%and22.08±4.54%respectively, P<0.05).3. Compared with the controls, the percentage of reticulated platelet of ITP patients were notablely enhanced (P<0.05). There was no apparent difference of the level of thrombopoietin between ITP patients with megakaryocyte cytosis and health controls (P>0.05), however, the level of thrombopoietin of ITP patients with normal megakaryocyte apparently increased (P<0.05).In patients with ITP, the count of platelets has negative correlation with the percentage of reticulated platelet and the level of thrombopoietin.Conclusion:1. Detecting the frequencies of Circulating B Cells Secreting Platelet-Specific Antibody in the patients with ITP, which can reflect the pathogenesis of ITP and have high sensitivity and specificity than modified MAIPA, could be able to improve the diagnosis and guide clinical therapy in some degree.2. Using the ELISPOT detecting the frequencies of Circulating B Cells Secreting Platelet-Specific Antibody in the patients with ITP could be able to discriminate the newly diagnosed and persistent and chronic ITP in some degree.3. Pathogenesis of ITP involves the disequilibrium of proportion of T lyphocyte subsets and the disorder of their function:To regulate the balance of T lymphocyte subsets may be a new approach to the treatment of ITP.4. There was no apparent difference of the level of thrombopoietin between ITP patients with megakaryocyte cytosis and health controls, however, the level of thrombopoietin of ITP patients with normal megakaryocyte apparently increased. The test of the concentration of TPO has important significance for the study of regulation mechanism of the platelet generation and the diagnosis of ITP.5. Reticulated platelet percentage in the serum of patients with ITP was obviously higher than that in healthy controls and there was inverse correlation between it and the platelet count. Therefore, reticulated platelet percentage can be the reference index for predicting the recovery state of the platelets in the treatment of ITP patients and has the important clinical significance in differentiating and diagnosing diseases caused by the decrease of platelets.