Functional characterization of annexin A5 gene promoter allelic variants

Background: M1 and M2 haplotypes are defined by 4 consecutive allelic variants in regulatory regions of the annexin A5 gene and have been found to reduce promoter activity. To date, no research has been carried out to investigate differential and individual impact each of the allelic variants has on promoter activity. In the current study, we functionally characterized the M1 and M2 haplotype allelic variants (c.-467G>A, c.-448A>C, c.-422T>C, c.-373G>A). We also characterized two other allelic variants located in the same regulatory region (c.-628C>T, c.-302T>G). Materials and methods: Their impact on the ANXA5 promoter activity was examined using a luciferase reporter assay in BeWo cells. Electrophoretic mobility shift assay with probes centered around each polymorphism was used to examine the binding ability of the allelic variants to nuclear proteins from BeWo cells. Results: Only the c.-467G>A and c.-628C>T allelic variants influenced the activity of the ANXA5 promoter, as measured by luciferase activity. Differential specific interactions with nuclear proteins were obtained for all allelic variants, except for the c.-302T>G, indicating that these polymorphisms could have an impact on the ANXA5 expression. Conclusions: We have functionally characterized allelic variants in the ANXA5 promoter, both alone and in combinations, and the results suggest that combinations of several individual variants contribute to modulate the ANXA5 transcriptional activity, most likely through binding of nuclear factors. These results provide new knowledge and insight into the mechanisms underlying the regulation of annexin A5 levels in healthy controls. (C) 2016 Elsevier Ltd. All rights reserved.