Clinicaland Molecular Characteristics Of Severe Congenital Neutropenia Caused By Elane Gene Mutations And The Unique Role Of The Hepatitis Virus B X Protein On HEK 293 Cell Morphology And Cellular Change

Part one : Clinical And Molecular Characteristics of Severe Congenital Neutropenia caused by ELANE Gene MutationsObjective: TO investigate the clinical feature, genetic and immunological characteristics of Severe Congenital Neutropenia(SCN)causing by ELANE gene mutationsMethods: We had investigated Eleven Chinese pediatric patients with suspected SCN. According to the Clinical data, such as peripheral blood,immune function,bone marrow ect. We had analyzed the PCR products from ELANE genomic DNA or c DNA and directly sequencedResults: The eleven patients all experienced typical recurrent infection such as pneumonia,oral cavity ulcer or soft tissue infection.Non-infectious symptoms such as neurologic deficits, osteopenia and anemia were found in some patients, and absolute peripheral neutrophil counts all varied. The eleven patients all have a different mutation sites according to results of DNA and c DNA sequencing,most patients harbored a range of heterozygous ELANE gene mutations, such as deletion,substitution and insertion caused frame shift alterations. Five mutations had not been reported previously;The PCR and direct sequencing results: P1 4exon F c.367-2 a>g splicing sites het mutation,may be affect RNA splicing,had not been reported previously;P1 5 exon F c.655G>A p.Val219 Ile had been reported previously;P1 Father 5 exon F c.655G>A p.Val219 Ile had been reported previously; P2 3 exon F c.366+10 splicing sites insert 13 bp AGAATCTCCAGGT had not been reported previously;P3 4 exon F c.368 T>A p.Leu123 His had not been reported previously;P45 exon F c.608G>C p.Gly203 Asp had been reported previously;P5 1exon F c.16c>a SNP had not been reported previously;P6 2 exon F c.125C>T p.Pro42 Leu had been reported previously;P6 4 exon F g.855322 C>A SNP had been reported previously;P7 4 exon F c.568 G>A p.Val190 Met had been reported previously;P8 5 exon F c.669 C>A p.Cys223 X had been reported previously;P9 4 exon F c.597 C>T p.F>F SNP had not been reported previously; P9 Father 4 exon F c.597 C>T p.F>F had not been reported previously; P9 4 exon F c.164 G>A p.Cys55 Thr had not been reported previously; 2 patients with CD40 L mutation:P10 CD40 L c.156+1 G>T splicing sites het mutation, had not been reported previously; P11 CD40 L c.156+1 G>T splicing sites het mutation, had not been reported previously.Conclusion: SCN cases were identified in China. Same patients carried novel ELANE mutations. Granulocyte-colony stimulating factor(G-CSF) was an effective treatment for most of the SCN patients.Granulocyte-colony stimulating factor(G-CSF) can prevented life-threatening or severe bacterial infections.Part two: The unique role of the hepatitis virus B X protein on HEK293 cell morphology and cellular changeObjective: To investigate The unique role of the hepatitis virus B X protein on HEK 293 cell morphology and cellular change. Illuminated same mechanisms of HBx regulated functions when comes to HEK 293cellMethods : Flow cytometry was used to detect the levels of PI associated with the nuclear DNA to determine the changes to the cellcycle.HEK 293 cells transfected with EGFP-N3(HEK 293) and EGFP-N3-HBx(HEK 293-HBx). Western blot of cell lysates from cells transfected with EGFP-N3(HEK 293) and EGFP-N3-HBx(HEK293-HBx). CDK4 antibody was used as a probe. Fluorescence images of cells transfected with EGFP-N3(HEK 293) and EGFP-N3-HBx(HEK293-HBx) to detect morphology change.Results: HBx increases cellular interaction by fusing the gaps between cells. HBx can repress the expression of E-cadherin at the transcriptional level, disrupting the association of E-cadherin with the actin cytoskeleton HBx enhances E-cadherin expression by increasing the expression level of Src in HEK 293 cells. HBx has been suggested to positively regulate the cell cycle HBx promotes the cell cycle by increasing the percentage of cells that are in division and expression of CDK4. HBx promotes cell proliferation and inhibits apoptosis by increasing Bcl2.Conclusion: The different functions of HBx in HEK 293 cells suggest that its role is cell dependent. HBx has been suggested to positively regulate the cell cycle HBx promotes the cell cycle by increasing the percentage of cells that are in division and expression of CDK4.