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

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.

Successful restoration of archived ovine formalin fixed paraffin-embedded tissue DNA and single nucleotide polymorphism analysis.

Archived formalin fixed paraffin-embedded (FFPE) tissues are powerful tools in medicine, capable of harboring diagnostic and genetic answers to challenging clinical questions. Successful utilization of DNA derived from FFPE samples is dependent upon repairing DNA damage generated from the fixation process. Methods to repair FFPE DNA have been successful in human medicine for a variety of research and clinical applications, yet remain underutilized in veterinary medicine. Despite the available technology, our study is the first to evaluate the repair of FFPE derived DNA from veterinary species for single-nucleotide polymorphism (SNP) analysis using the Illumina OvineSNP50 BeadChip and Illumina FFPE QC and DNA Restore kit. To accomplish this, 48 ovine FFPE samples were run using the Illumina OvineSNP50 BeadChip with and without restoration. Compared to pre-restore data, we found increased sample call rates, SNP call frequency, and assay metrics for all samples post-restoration. Further, we utilized four sheep with available parallel fresh DNA and FFPE DNA to compare assay metrics and genotype calls between the two starting sample types. Although fresh samples generated increased call rates, we found 99% concordance in allele calls between restored FFPE and fresh DNA for all four samples. Our results indicate successful restoration and genotyping of ovine FFPE samples using this technology, with potential for utilization in other veterinary species.

The Paratuberculosis Paradigm Examined: A Review of Host Genetic Resistance and Innate Immune Fitness in Mycobacterium avium subsp. Paratuberculosis Infection.

Paratuberculosis, or Johne's Disease (JD) is a debilitating chronic enteritis mainly affecting ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This organism causes worldwide economic losses to the livestock industry, and is of public health importance due to the potential zoonotic risk between MAP and Crohn's disease (CD) in humans. Without economical treatments, or a vaccine capable of preventing infection without causing cross-reactions with bovine tuberculosis, test-and-cull methods for disease control are imperative. Unfortunately, difficulties in diagnostics and long subclinical stage hinder adequate control and is further complicated by variation in MAP exposure outcome. Interestingly, the majority of infections result in asymptomatic presentation and never progress to clinical disease. One contributing factor is host genetics, where polymorphisms in innate immune genes have been found to influence resistance and susceptibility to disease. Candidate genes identified across studies overlap with those found in CD and tuberculosis including; Solute carrier family 11 member 1 gene (SLC11A1), Nucleotide-binding-oligomerization domain containing gene 2 (NOD2), Major histocompatibility complex type II (MHC-II), and Toll-like receptor (TLR) genes. This review will highlight evidence supporting the vital role of these genes in MAP infection outcome, associated challenges, and implications for the future of JD research.

Multiparametric MRI for early identification of therapeutic response in recurrent glioblastoma treated with immune checkpoint inhibitors.

BACKGROUND: Physiologic changes quantified by diffusion and perfusion MRI have shown utility in predicting treatment response in glioblastoma (GBM) patients treated with cytotoxic therapies. We aimed to investigate whether quantitative changes in diffusion and perfusion after treatment by immune checkpoint inhibitors (ICIs) would determine 6-month progression-free survival (PFS6) in patients with recurrent GBM. METHODS: Inclusion criteria for this retrospective study were: (i) diagnosis of recurrent GBM treated with ICIs and (ii) availability of diffusion and perfusion in pre and post ICI MRI (iii) at ≥6 months follow-up from treatment. After co-registration, mean values of the relative apparent diffusion coefficient (rADC), Ktrans (volume transfer constant), Ve (extravascular extracellular space volume) and Vp (plasma volume), and relative cerebral blood volume (rCBV) were calculated from a volume-of-interest of the enhancing tumor. Final assignment of stable/improved versus progressive disease was determined on 6-month follow-up using modified Response Assessment in Neuro-Oncology criteria. RESULTS: Out of 19 patients who met inclusion criteria and follow-up (mean ± SD: 7.8 ± 1.4 mo), 12 were determined to have tumor progression, while 7 had treatment response after 6 months of ICI treatment. Only interval change of rADC was suggestive of treatment response. Patients with treatment response (6/7: 86%) had interval increased rADC, while 11/12 (92%) with tumor progression had decreased rADC (P = 0.001). Interval change in rCBV, Ktrans, Vp, and Ve were not indicative of treatment response within 6 months. CONCLUSIONS: In patients with recurrent GBM, interval change in rADC is promising in assessing treatment response versus progression within the first 6 months following ICI treatment. KEY POINTS: • In recurrent GBM treated with ICIs, interval change in rADC suggests early treatment response.• Interval change in rADC can be used as an imaging biomarker to determine PFS6.• Interval change in MR perfusion and permeability measures do not suggest ICI treatment response.