A Functional Variant in the Promoter Region of EDN2 Associated with Map Tissue Infection Up-Regulates EDN2 Expression

Johne’s disease is a transmissible bacterial disease in cattle caused by Mycobacterium avium subspecies paratuberculosis (Map) infection.  A GWAS undertaken by our research group identified 16 SNPs in a 70 kb region on BTA3 as being associated (P<0.01) with Map tissue infection.   This study’s objective was to investigate 3 EDN2 SNPs to determine if one or more was a causal variant for Map tissue infection.  The EDN2 SNPs investigated were SNP 272 (G/A at +5738 bp) in the 3’UTR and two SNPs in the EDN2 promoter (SNP 105 (A/G) at -11kb and SNP 208 (G/T) at -671 bp). Nuclear extracts from freshly harvested ileo-cecal nodes were used for microRNA (miRNA) and electromobility shift assays (EMSA) using the NE-PER kit (Thermo Scientific, Rockford, IL). All assays were conducted in triplicate. Interference assays for the microRNA binding sites (miR-1197 and miR-2339) for SNP 272 showed no effect on altering EDN2’s stability or expression.   EMSAs for SNP 105 and SNP 208 demonstrated differential binding affinity to nuclear proteins dependent on the 105 (>400 fold change between A and G alleles) and 208 (>100 fold change between G and T alleles) SNP alleles.  A luciferase reporter assay confirmed that transcriptional activity of the EDN2 promoter was increased 2 fold (P=0.001, coefficient variance 7%) with the G allele for SNP 208.  Reporter assays for SNP 105 are underway.   These results suggest that at least one variant in the promoter of EDN2 may serve as a causal variant for susceptibility to Map tissue infection.