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

Abstract

Background: Acute myocardial infarction (AMI), a common complex disease caused by an interaction between genetic and environmental factors, is a serious type of coronary artery disease and is also a leading cause of death worldwide. Autophagy-related 16-like 1 (ATG16L1) is a key regulatory factor of autophagy and plays an important role in induced autophagy. In the cardiovascular system, autophagy is essential to preserve the homeostasis and function of the heart and blood vessels. No studies have hitherto examined the association between AMI and ATG16L1 gene promoter.

Methods: We conducted a case-control study, using polymerase chain reaction and sequencing techniques, dual luciferase reporter assay, and electrophoretic mobility shift assay, to analyze genetic and functional variation in the ATG16L1 gene promoter between AMI and controls. A variety of statistical analyses were used to analyze the allele and genotype frequencies and the relationship between single-nucleotide polymorphisms (SNPs) and AMI.

Results: In all, 10 SNPs and two DNA-sequence variants (DSVs) were identified in 688 subjects, and three ATG16L1 gene promoter mutations [g.233250693 T > C (rs185213911), g.233250946 G > A (rs568956599), g.233251133 C > G (rs1301744254)] that were identified in AMI patients significantly altered the transcriptional activity of ATG16L1 gene promoter in HEH2, HEK-293, and H9c2 cells (P < 0.05). Further electrophoretic mobility shift assays indicated that the SNPs affected the binding of transcription factors (P < 0.01).

Conclusion: ATG16L1 gene promoter mutations in AMI patients may affect the binding of transcription factors and change the transcriptional activity of the ATG16L1 gene, changing the level of autophagy and contributing to the occurrence and development of AMI as rare and low-frequency risk factors.

Keywords: ATG16L1; acute myocardial infarction; autophagy; gene promoter; single-nucleotide polymorphisms.

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.