Retrospective Single Nucleotide Polymorphism Analysis of Host Resistance and Susceptibility to Ovine Johne's Disease Using Restored FFPE DNA

Abstract

Johne's disease (JD), also known as paratuberculosis, is a chronic, untreatable gastroenteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. Evidence for host genetic resistance to disease progression exists, although it is limited due to the extended incubation period (years) and diagnostic challenges. To overcome this, previously restored formalin-fixed paraffin embedded tissue (FFPE) DNA from archived FFPE tissue cassettes was utilized for a novel retrospective case-control genome-wide association study (GWAS) on ovine JD. Samples from known MAP-infected flocks with ante- and postmortem diagnostic data were used. Cases (N = 9) had evidence of tissue infection, compared to controls (N = 25) without evidence of tissue infection despite positive antemortem diagnostics. A genome-wide efficient mixed model analysis (GEMMA) to conduct a GWAS using restored FFPE DNA SNP results from the Illumina Ovine SNP50 Bead Chip, identified 10 SNPs reaching genome-wide significance of p < 1 × 10^-6 on chromosomes 1, 3, 4, 24, and 26. Pathway analysis using PANTHER and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was completed on 45 genes found within 1 Mb of significant SNPs. Our work provides a framework for the novel use of archived FFPE tissues for animal genetic studies in complex diseases and further evidence for a genetic association in JD.

Keywords: DNA repair; FFPE tissues; GWAS; host susceptibility; paratuberculosis.

Figure 1

Manhattan plot p < 1 × 10⁻⁶ showing the distribution of the 10 significant SNPs identified above the calculated genome-wide significance threshold of p < 1 × 10⁻⁶. Figure generated using the RStudio Package qqma. Each chromosome is located on the x-axis, and statistical significance on y-axis, each individual SNP is represented as a single dot along a given chromosome.

Figure 2

Q-Q Plot: Observed vs. expected p-values are plotted and were made in the RStudio Package: qqma. Showing expected vs. observed is a graphical representation of the deviation of the observed p-values from the null hypothesis indicating few SNPs are in linkage-disequilibrium with a causal polymorphism resulting in small p-values.