Genetic Variants and Functional Analyses of the ATG16L1 Gene Promoter in Acute Myocardial Infarction

Figures

• 

  FIGURE 1. Schematic diagram of gene mutation site of ATG16L1 gene promote. The DSVs and SNPs in the ATG16L1 gene promoter identified in AMI patients and controls. Schematic representation of the DSVs within the ATG16L1 gene promoter. The numbers represent the genomic DNA sequences of the human ATG16L1 gene (Genbank accession number NC_000002.12) upstream to the transcription start site (+1).

• 

  FIGURE 2. Sequencing chromatograms of the DSVs and SNPs within the ATG16L1 gene promoter in AMI patients and controls. All sequence orientations of the DSVs and SNPs are forward. (A) Sequencing chromatograms of the SNPs in AMI patients. (B) Sequencing chromatograms of the DSVs and SNP in healthy controls. The top layers are wild-type sequences, the bottom panels are heterozygous mutations, and the mutation sites are marked with arrows.

• 

  FIGURE 3. The LD tests of the Nine SNPs in AMI. Standard color schemes indicate different levels of LD. Standard Color Scheme: White: LOD < 2, D′ < 1; Blue: LOD < 2, D′ = 1; Bright red, LOD > 2, D′ = 1; LOD: the log of the likelihood odds ratio, is a measure of confidence in the value of D′. Shown LD value: R-squared. The box is empty indicates that the r2 value is 1.000.

• 

  FIGURE 4. Relative transcriptional activity of wild-type and variant ATG16L1 gene promoters. Wild-type and variant ATG16L1 gene promoters were cloned into reporter gene vector pGL3 and transfected into cultured cells. The transfected cells were collected, and dual-luciferase activities were assayed. Empty vector pGL3-basic was used as a negative control. The transcriptional activity of the wild-type ATG16L gene promoter was designed as 100%. The relative activities of the ATG16L1 gene promoter were calculated. Relative activities of wild-type and variant ATG16L1 gene promoters in HEK-293, H9c2 and HEH2 cells. Lanes 1, pGL3-basic; 2, pGL3-WT; 3, pGL3-233250693C; 4, pGL3-233250873A; 5, pGL3-233250946A; 6, pGL3-233251039C; 7, pGL3-233251111T; 8, pGL3-233251133G; 9, pGL3-233251186G; 10, pGL3-233251524C; 11, pGL3-233251563T, and 12, pGL3-233251699G. **p < 0.01; ***p < 0.001.

• 

  FIGURE 5. EMSA of SNPs biotin-labeled oligonucleotides in AMI patients. Wild-type and mutant oligonucleotides (30 bp) were designed, and SNPs of AMI patients were labeled with biotin, including [g.233250693 T>C (rs185213911), g.233250946 G>A (rs568956599), and g.233251133 C>G (rs1301744254)]. EMSA was conducted with biotinylated oligonucleotides and the nuclear extracts from HEK-293 and H9c2 cells. The free probe was marked with an arrow at the bottom. The affected binding for transcription factors was marked with an open arrow.