Research Article

Raet1e Polymorphisms Are Associated with Increased Risk of Developing Premature Coronary Artery Disease and with Some Cardiometabolic Parameters: The GEA Mexican Study

Figure 1

rs7756850 affects transcriptional activity of raet1e promoter sequence. (a) Schematic diagram of the raet1e gene and location of allelic variants on promoter sequence (−426 bp, G/C). (b) Construction of plasmid. The promoter sequence with each variant was cloned in the pGL3 vector into MluI/XhoI sites to direct the expression of the luciferase gene. Both constructions (G allele and C allele) were confirmed by sequencing. (c) Dual-luciferase assay in HEK293 cells. The presence of the C allele reduces the promoter activity of the cloned sequence (−503 bp) in ≈85% as compared with the promoter activity of the sequence harboring the G allele (). Lines represent the median of four independent experiments performed in triplicate (). was determined by the Mann-Whitney test. (d) In silico analysis suggests that variant rs7756850 modifies the consensus site for p53 and Nkx2.5 transcription factors. Due to the raet1e gene orientation into chromosome 6, the G allele is considered the ancestral allele in the luciferase assays.