The Role Of BRIP1 Mutations In Acute Myeloid Leukemia

ObjectiveTo detect the mutation of BRIP1 gene in adult acute myeloid leukemia(AML)patients.We analyzed the role of BRIP1 mutation in the prognosis of AML patients.The study aimed to explore the molecular mechanism of BRIP1 gene mutation in tumorigenesis.Method1.This study amplified the BRIP1 gene by PCR method and sequenced the gene using Sanger strategy in 241 AML patients.The cDNAs were extracted and transcripted from bone marrow cells and normal skin cells.2.All statistical analyses were performed using SPSS 20.0 statistical software.Kaplan-Meier method and Cox regression model were used for univariate and multivariate analysis.All statistics were 2-tailed.P values below 0.05 were considered statistically significant.3.Mutation sites were blasted in different organisms to analyze the evolutionary conservation status.4.We predicted the structure model of BRIP1 protein The BRCA1-binding domain model was built from PDB 4v1a_P.The helicase domain was built from PDB 4a15.5.Human BRIP1 gene was cloned in plvx-EF1-MCS-PGK-PURO expression vector.The BRIP1 missense mutations were generated by Q5(?)Site-Directed Mutagenesis Kit from NEB company BRIP1 proteins(both wild-type and mutants)were overexpressed in HEK293T cell and recombinant proteins were purified by immunoprecipitation and competing elution methods.The DNA helicase activities of proteins were detected by fluorescent labeling method.6.Construct BRIP1 genes(both wild-type and mutants)over-expressed 32D cell line.We examined cell survival and apoptosis after DNA damage treatment.Result1.There were 11 mutation sites in the coding region of BRIP1 gene,namely,A144T,N196S,R419W,G481D,L537V,P787L,R814C,H870Y,1896V,Q944E,D1138N.2.There was a statistical difference in the prognosis between the mutant group and the wild-type group.3.Homologous comparison showed that mutation sites of A144,R419,G481,L537,P787,H870,Q944 and D1138 were highly conserved in different organisms.4.Prediction of BRIP1 protein structure model showed that mutation sites of G481,R814,H870,Q944 and other sites may be located in the BRIP1 protein's important functional structure area.5.BRIP1 genes(both wild-type and mutants)were successf?lly cloned in plvx-EF1-MCS-PGK-PURO vector,and proteins were successfully expressed and purified by WB sliver staining detection.The activities of BRIP1 proteins(both wild-type and mutants)were different by fluorescent labeling method.6.The cell survival and apoptosis of different mutanat cell lines were found to be different.ConclusionSuccessfully constructed the BRIP1 expression plasmids,and purified the proteins from HEK293T cells,in the meanwhile,found that the activities of BRIP1 wild-type and mutated proteins were different.