Analysis Of Promoter Ploymorphism Of BRCA2in Association With Breast Cancer Prognosis And Cloning And Analysis Of The BRCA2Promoter

Part1. Analysis of promoter polymorphism of BRCA2in associationwith breast cancer prognosisObjective: To analysis the functional polymorphism in the promoter region of theBRCA2gene with breast cancer prognosis.Methods: We performed study in491breast cancer cases to test the associationbetween four polymorphisms and breast cancer prognosis.Genotypes were determinedin DNA from blood cells by direct sequencing.Results: The result showed that-1144C/T polymorphism was associated withclinical outcome. Carriers of the TT genotype had a longer disease free survival(p=0.029). The result was more evident among sporadic breast cancer patients withunilateral cancer (p=0.010). Linkage disequilibrium (LD) analysis showed that all thefive SNPs are in Linkage disequilibrium (D’>0.8).Conclusions: This study showed that the-1144C/T polymorphism in BRCA2genepromoter may affect prognosis of breast cancer in Chinese. Its significance in otherpopulations remains to be investigated. Part2:Cloning and analysis of the BRCA2promoterObjective: To construct a BRCA2promotor luciferase reporter gene vector and toanalyze the mechanism of the gene regulation.Methods: By using genomic DNA from healthy people an a template, the BRCA2promotor region was amplified by high fidelity DNA polymerase. Then the fragmentwas cloned into a recombinant plasmid. To analyze whether polymorphismrs3092989(-254A/G) could affect promoter activity, PCR-based mutagenesis wasperformed to generate haplotype containing-254G allele. Affer confirmed by directsequencing, the BRCA2promotor was subclonged into pGL3-basic vector. Theconstructed vector was transfected into plasmid HeLa cells to detect promoter activity.Results: Direct sequencing and restriction endonuclease analysis confirmed therecombinant plasmid contained pGL3-basic vector and BRCA2promoter sequence. TheBRCA2promoter containing-254A and-254G allele was successfully cloned. TheBRCA2promoter exhibited strong promoter activity with an increase of413fold ofrelative luciferase activity unit (RLU) as compared with pGL3-basic vector.The-254A/G polymorphism did not affect promoter activity.Conclusions: The cloned BRCA2promoter region has strong promoteractivity.The success of cloning and mutagenesis of BRCA2promoter region could beused for further analysis of gene regulation.