Preliminary Study Of New Pathogenic Mutations In Schimke Immuno-osseous Dysplasia

Objective:Construction of wild and mutant SMARCAL1 lentiviral vectors and preliminary study on the effect of new pathogenic mutations of Schimke immuno-osseous dysplasia?SIOD?on the expression of SMARCAL1 protein.Methodology:A peripheral blood sample of a newly diagnosed SIOD patient was collected for gene sequencing,and new mutation sites were detected by detection.The protein analysis software was used to predict the function of the new mutant protein.The wild-type and mutant SMARCAL1 gene sequences were synthesized by PCR,and the mutant SMARCAL1 gene sequence contained the new mutation site.The vector pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO was prepared,and a linearized vector was obtained by a digestion reaction.Primers containing a cleavage site were designed,and wild-type and mutant SMARCAL1 gene fragments were obtained by PCR amplification technique.The linearized vector and the target gene fragment were ligated and recombined by HB-infusion^TM seamless cloning kit.The recombinant plasmid product was added to the competent cell DH5?suspension for transformation,single colony of single plate was picked for PCR identification,and positive clone transformants were selected for sequencing analysis.The wild-type recombinant transformants and the mutant recombinant transformants with correct sequence were subjected to plasmid extraction separately,then,the extracted plasmid was co-infected with 293T cells with the lentiviral packaging helper plasmid,and the virus supernatant was collected twice at 48 hours and 72 hours after transfection,purified by ultracentrifugation,and packaged after determination of virus titer.HEK293 cells were sub-cultured.The optimal Polybrene concentration and optimal MOI value of HEK293cells transfected with lentivirus were studied through preliminarily experiments.HEK293cells were transfected with empty-type,wild-type and mutant lentiviral vectors,respectively,and screened for 3 generations with appropriate concentration of puromycin to obtain three stable cell lines.The total protein of normal HEK293 cells with empty-type,wild-type and mutant stable cell lines were extracted,then,the expression of SMARCAL1 protein in four groups was detected by Western Blot.Results:Genetic testing of child with SIOD revealed the SMARCAL1 gene?NM₀14140.3?Exon5:c.1071delT;p.?Phe357fs?.This mutation is a frameshift mutation that is predicted to be deleterious by protein analysis software and is expected to result in a truncation of the protein encoded by the SMARCAL1 gene,thereby affecting his normal biological function.No reports of this mutation were found in the databases of HGMD,ESP6500siv2_ALL,Thousand Human Genome and dbSNP147.By sequencing the wild-type and mutant positive clone transformants separately and analyzing the wild-type and mutant SMARCAL1 gene fragment sequences,it was confirmed that they were consistent with the wild-type and mutant target region sequences,suggesting that the wild-type and mutant-type SMARTAR1 were successfully constructed.After 48 hours of lentivirus transfection into HEK293 cells,green fluorescence was observed under a fluorescence microscope,suggesting successful transfection.Western Blot results showed that the expression level of SMARCAL1 protein in the mutant group was significantly lower than that in the wild group,normal group and empty group,P<0.05;suggesting that the new mutation caused the decrease of SMARCAL1 protein expression.Conclusion:The peripheral blood gene test of the child found a new frameshift mutation in SMARTAL1,and the protein analysis software predicted that the frameshift mutation would cause the protein encoded by the SMARCAL1 gene to be truncated and affect its normal biological function.Wild-type and mutant SMARCAL1 overexpressing lentiviral vectors were successfully constructed for subsequent in vitro cell experiments.HEK293 cells were successfully transfected to obtain wild-type and mutant SMARCAL1stably transfected cell lines.Western Blot confirmed that the new mutation of SMARTAL1 was a SIOD pathogenic mutation.