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).


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.
Atherosclerosis and Thrombosis Unit, IRCCS Casa Sollievo della Sofferenza Hospital, 71013 San Giovanni Rotondo, Italy.
Giovanni Luca
GL
Tiscia
Department of Haematology, Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, 0424 Oslo, Norway.
Elisabeth
E
Dørum
Department of Haematology, Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, 0424 Oslo, Norway.
Christiane Filion
CF
Myklebust
Atherosclerosis and Thrombosis Unit, IRCCS Casa Sollievo della Sofferenza Hospital, 71013 San Giovanni Rotondo, Italy.
Elvira
E
Grandone
Department of Haematology, Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, 0424 Oslo, Norway; Institute of Clinical Medicine, University of Oslo, 0370 Oslo, Norway.
Per Morten
PM
Sandset
Atherosclerosis and Thrombosis Unit, IRCCS Casa Sollievo della Sofferenza Hospital, 71013 San Giovanni Rotondo, Italy. Electronic address: grethe.skretting@medisin.uio.no.
Grethe
G
Skretting
D000483:Alleles; D017304:Annexin A5 / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D005786:Gene Expression Regulation; D006239:Haplotypes; D006801:Humans; D010920:Placenta / Q000166:cytology; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011401:Promoter Regions, Genetic
D017304:Annexin A5
Alleles; Electrophoretic mobility shift assay; Gene expression regulation; Genes, reporter; Promoter regions